Isolation, cultivation and uses of stem/progenitor cells

ABSTRACT

The present invention relates to a method for isolating stem/progenitor cells from the amniotic membrane of umbilical cord, wherein the method comprises separating the amniotic membrane from the other components of the umbilical cord in vitro, culturing the amniotic membrane tissue under conditions allowing cell proliferation, and isolating the stem/progenitor cells from the tissue cultures. The isolated stem cell cells can have embryonic stem cell-like properties and can be used for various therapeutic purposes. In one embodiment, the invention relates to the isolation and cultivation of stem cells such as epithelial and/or mesenchymal stem/progenitor cells under conditions allowing the cells to undergo mitotic expansion. Furthermore, the invention is directed to a method for the differentiation of the isolated stem/progenitor cells into epithelial and/or mesenchymal cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 11/205,248filed Aug. 15, 2005, which claims the benefit of priority of U.S.Provisional Application No. 60/602,208, filed Aug. 16, 2004, U.S.Provisional Application No. 60/632,209, filed Dec. 1, 2004 andInternational Application No. PCT/SG2005/000174, filed Jun. 3, 2005, thecontents of each being hereby incorporated by reference it its entiretyfor all purposes.

FIELD OF THE INVENTION

The present invention relates to a method for isolating stem/progenitorcells from the amniotic membrane of umbilical cord, wherein the methodcomprises separating the amniotic membrane from the other components ofthe umbilical cord in vitro, culturing the amniotic membrane tissueunder conditions allowing cell proliferation, and isolating thestem/progenitor cells from the tissue cultures. In particular, theinvention relates to the isolation and cultivation of stem cells havingembryonic properties such as epithelial and/or mesenchymalstem/progenitor cells under conditions allowing the cells to undergomitotic expansion. Furthermore, the invention is directed to a methodfor the differentiation of the isolated stem/progenitor cells intoepithelial and/or mesenchymal cells and therapeutic uses of thesestem/progenitor cells.

BACKGROUND OF THE INVENTION

Stem cells are a cell population possessing the capacities to self-renewindefinitely and to differentiate in multiple cell or tissue types.Embryonic stem cells (from approximately days 3 to 5 afterfertilisation) proliferate indefinitely and can differentiatespontaneously into all tissue types: they are thus termed pluripotentstem cells (reviewed, for example, in Smith, A. G. (2001) Annu. Rev.Cell. Dev. Biol. 17, 435-462). Adult stem cells, however, are moretissue-specific and may have less replicative capacity: they are thustermed multipotent stem cells (reviewed, for example, in Paul, G. et al.(2002) Drug Discov. Today 7, 295-302). The “plasticity” of embryonic andadult stem cells relies on their ability to trans-differentiate intotissues different from their origin and, perhaps, across embryonic germlayers.

The ability of stem cells to self-renew is critical to their function asreservoir of primitive undifferentiated cells. In contrast, most somaticcells have a limited capacity for self-renewal due to telomereshortening (reviewed, for example, in Dice, J. F. (1993) Physiol. Rev.73, 149-159). Stem cell-based therapies thus have the potential to beuseful for the treatment of a multitude of human and animal diseases.

Stem cells as well as stem/progenitor cells can be derived fromdifferent sources. The “multi-lineage” potential of embryonic and adultstem cells has been extensively characterized. Even though the potentialof embryonic stem cells is enormous, their use implies many ethicalproblems. Therefore, non-embryonic stem cells derived from the bonemarrow stroma, fat tissue, dermis and umbilical cord blood have beenproposed as alternative sources. These cells can differentiate interalia into chondrocytes, adipocytes, osteoblasts, myoblasts,cardiomyocytes, astrocytes, and tenocytes in vitro and undergodifferentiation in vivo, making these stem cells—in general referred toas mesenchymal stem cells—promising candidates for mesodermal defectrepair and disease management.

In clinical use, however, harvesting of such mesenchymal stem cellscauses several problems. The collection of the cells is a mental andphysical burden to the patient as a surgical procedure is required toobtain the cells (for example, the collection of bone marrow is aninvasive technique performed with a biopsy needle that requires local oreven general anesthesia). Furthermore, in many cases the number of stemcells extracted is rather low. More importantly, no epithelial cells arederived or differentiated from these cells. This prompted the search forother possible sources of stem cells.

Umbilical cord blood has been identified as a rich source ofhaematopoetic stem/progenitor cells. However, the existence ofmesenchymal stem/progenitor cells is discussed controversially. On theone hand, such cells could not be isolated or successfully cultured fromterm umbilical cord blood (Mareschi, K. et al. (2001) Haematologica 86,1099-1100). At the same time, results obtained by Campagnoli, C. et al.(Blood (2001) 98, 2396-2402) as well as Erices, A. et al. (Br. J.Haematol. (2000) 109, 235-242) suggest that mesenchymal stem cells arepresent in several fetal organs and circulate in the blood of pre-termfetuses simultaneously with hematopoietic precursors. Accordingly,International Patent Application WO 03/070922 discloses isolation andculture-expansion methods of mesenchymal stem/progenitor cells fromumbilical cord blood and a differentiation method of such cells intovarious mesenchymal tissues. Isolation efficiencies of about 60% havebeen reported (Bieback, K. et al. (2004) Stem Cells 22, 625-634). In thesame study, both the time period from collection of the umbilical cordblood to isolation of the cells and the volume of the blood sample usedhave been determined as crucial parameters for achieving such a yield.However, it is still a matter of debate whether these stem/progenitorcells are indeed derived of umbilical cord tissue.

Recently, mesenchymal stem/progenitor cells have been successfullyisolated from umbilical cord tissue, namely from Wharton's jelly, thematrix of umbilical cord, (Mitchell, K. E. et al. (2003) Stem Cells 21,50-60; U.S. Pat. No. 5,919,702; US Patent Application 2004/0136967).These cells have been shown to have the capacity to differentiate, forexample, into a neuronal phenotype and into cartilage tissue,respectively. Furthermore, mesenchymal stem/progenitor cells have alsobeen isolated from the endothelium and the subendothelial layer of theumbilical cord vein, one of the three vessels (two arteries, one vein)found within the umbilical cord (Romanov, Y. A. et al. (2003) Stem Cells21, 105-110; Covas, D. T. et al. (2003) Braz. J. Med. Biol. Res. 36,1179-1183).

However, none of these approaches employed thus far has resulted in theisolation or cultivation of epithelial stem/progenitor cells as a sourcefor epithelial cell-based therapies such as skin resurfacing, liverrepair, bladder tissue engineering and other engineered surface tissues.Thus, there is still a need for methods and reliable sources useful forthe isolation and cultivation of epithelial stem/progenitor cells.Furthermore, rapid and efficient methods which are ethically acceptableand do not pose a biomedical burden on the patient for the isolation ofepithelial and mesenchymal stem/progenitor cells are still required inorder to provide such cells in a sufficient amount for variousapplications in regenerative medicine and tissue engineering.

SUMMARY OF THE INVENTION

The invention provides a method for isolating stem/progenitor cells fromthe amniotic membrane of umbilical cord, the method comprising:

-   -   (a) separating the amniotic membrane from the other components        of the umbilical cord in vitro;    -   (b) culturing the amniotic membrane tissue obtained in step (a)        under conditions allowing cell proliferation; and    -   (c) isolating the stem/progenitor cells.

In one embodiment, the invention provides a method, further comprising:

-   -   (a″) separating the cells from the amniotic membrane tissue        before cultivation by a technique selected from the group        consisting of enzymatic digestion and direct tissue explant.

In one preferred embodiment, the invention provides a method forisolating stem/progenitor cells that have embryonic stem cell-likeproperties.

In another preferred embodiment, the invention provides a method forisolating epithelial and/or mesenchymal stem/progenitor cells.

In another embodiment, the invention provides a method furthercomprising:

-   -   (d) culturing the stem/progenitor cells under conditions        allowing the cells to undergo clonal expansion.

In yet another embodiment, the invention provides a method furthercomprising:

-   -   (e) culturing the stem/progenitor cells under conditions        allowing the differentiation of said cells into epithelial cells        and/or mesenchymal cells; and    -   (f) isolating the differentiated cells.

In yet another embodiment, the invention provides a method, furthercomprising:

-   -   (g) preserving the isolated stem/progenitor cells for further        use.

In yet a further embodiment, the invention comprising a method ofcultivating stem/progenitors cells of the invention, comprising:

Obtaining a tissue explant from the amniotic membrane of umbilical cord;

Cultivating the tissue explant in suitable cultivation media andcultivation conditions over a suitable period of time.

In yet other embodiments, the invention is directed to therapeutic usesof the stem/progenitor cells or cells differentiated therefrom orcellular secretions or extracts thereof. One of these embodimentsprovide a method of treating a subject having a disorder comprisingadministering to the subject an effective amount of a stem/progenitorcell isolated by the inventive method of explained above. Anotherembodiment comprises administering to the subject an effective amount ofa cell differentiated from a stem/progenitor cell of the invention.Other embodiments provide a corresponding pharmaceutical composition,i.e. a pharmaceutical composition comprising a stem progenitor cell or acell differentiated therefrom, as well as cellular secretions into thecell medium and extracts.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood with reference to the detaileddescription when considered in conjunction with the non-limitingexamples and the drawings, in which:

FIG. 1 depicts epithelial cell outgrowth from umbilical cord amnioticmembrane by the method of direct tissue explant (40×magnification) atday 2 (FIG. 1A) and day 5 (FIG. 1B, C) of tissue culture. Cell cultureplastic surfaces were coated with collagen 1/collagen 4 mixtures (1:2;Becton Dickinson) before placing the amniotic membrane on the surface.The amniotic membrane specimens were submerged in 5 ml EpiLife medium orMedium 171 (both from Cascade Biologics). Medium was changed every 2 or3 days and cell outgrowth by explant was monitored under lightmicroscopy. Microphotographs were taken at different time intervals asstated above. The observed polyhedral cell morphology is typical ofepithelial cells.

FIG. 2 depicts enzymatic digestion of the umbilical cord segmentsyielding similar epithelial (40×magnification) cells at day 2 (FIG. A,C) and day 5 (FIG. B, D). Umbilical cord amniotic membrane was dividedinto small pieces of 0.5 cm×0.5 cm and digested in 0.1% (w/v)collagenase type 1 solution (Roche Diagnostics) at 37° C. for 8 hours.The samples were vortexed every 30 min for 3 min. Cells were harvestedby centrifugation at 4000 rpm for 30 min. Cell pellets were resuspendedin EpiLife medium or Medium 171 (both from Cascade Biologics)supplemented with 50 μg/ml insulin-like growth factor-1 (IGF-1), 50μg/ml platelet-derived growth factor-BB (PDGF-BB), 5 μg/ml transforminggrowth factor-β1 (TGF-β1) and 5 μg/ml insulin (all obtained from R&DSystems), counted and seeded on 10 cm tissue culture dishes pre-coatedwith collagen 1/collagen 4 mixtures (1:2; Becton Dickinson) at densityof 1×10⁶ cells/dish. After 24 hours, attached cells were washed withwarm phosphate buffered saline (PBS) and the culture medium was replacedwith EpiLife medium or Medium 171 (both from Cascade Biologics). Themedium was changed every 2 or 3 days, and cell outgrowth was monitoredunder light microscopy. Microphotographs were taken at different timeintervals as stated above. Once again the cells demonstrated typicalepithelial cell polyhedral morphology.

FIG. 3 depicts outgrowing mesenchymal cells explanted from umbilicalcord amniotic membrane. Cellular outgrowth was observed as early as 48hours after placement in tissue culture dishes using DMEM supplementedwith 10% fetal calf serum (FCS) as culture medium (40×magnification)(FIG. 3A, C). The explants were submerged in 5 ml DMEM (Invitrogen)supplemented with 10% fetal bovine serum (Hyclone) (DMEM/10% FBS).Medium was changed every 2 or 3 days. Cell outgrowth was monitored underlight microscopy. Microphotographs were taken at different timeintervals. The cells were characterized by their spindle shapedmorphology, and migrated and expanded both easily and quickly in vitro,closely resembling fibroblasts (FIG. 3B, D).

FIG. 4 (40×magnification) depicts mesenchymal cells from umbilical cordamniotic membrane cells isolated by collagenase enzymatic digestion.FIG. 4A shows mesenchymal cells isolated from umbilical cord amnioticmembrane at day 2. Cell proliferation was observed at day 5 (FIG. 4B).Umbilical cord amniotic membrane was divided into small pieces of 0.5cm×0.5 cm and digested in 0.1% (w/v) collagenase type1 solution (RocheDiagnostics) at 37° C. for 6 hours. The samples were vortexed every 15min for 2 min. Cells were harvested by centrifugation at 4000 rpm for 30min. Cell pellets were resuspended in DMEM/10% FBS, counted and seededon 10 cm tissue culture dish at density of 1×10⁶ cells/dish. Medium waschanged every 2 or 3 days. Cell outgrowing was monitored under lightmicroscopy. Microphotographs were taken at different time intervals.Once again, cells demonstrated spindle shaped morphology typical ofmesenchymal cells as fibroblasts.

FIG. 5 (40×magnification) depicts the morphology in serum-free culturecondition (DMEM) and serum culture condition (DMEM/10% FCS) of umbilicalcord amniotic membrane mesenchymal cells (UCMC, FIG. 5E, F, G, H)isolated according to the method of the invention, normal dermalfibroblasts (NF109 cells, FIG. 5A, B) and adipose-derived mesenchymalcells (ADMC, FIG. 5C, D). FIG. 5 shows changes in cell morphology of NFand ADMC cultured in serum starvation conditions (DMEM only) reflectedby flatter cells and less dense cytoplasm as compared with serum richconditions (DMEM/10%FCS) where cells are more rounded with a densecytoplasm (FIG. 5A, B, C, D). No change in morphology was observed inboth UCMC groups cultured under identical conditions of serum-free vs.serum rich media (FIG. 5E, F, G, H), indicating a difference in behaviorand physiology of these latter mesenchymal cells.

FIG. 6 (40×magnification) depicts UCMC isolated according to theinvention cultured in DMEM/10% FCS at days 3 and 7 without a 3T3 feederlayer. The cells are seen to be growing well, and are forming a colony(vertical growth) instead of exhibiting radial spread. Once again, thisindicates a difference in behavior of these mesenchymal cells ascompared to their more differentiated counterparts.

FIG. 7 (40×magnification) depicts colony formation of umbilical cordepithelial cells (UCEC) cultured on a 3T3 feeder layer at days 3 and 7.This appearance is similar to that of normal skin derived epithelialkeratinocyte stem cells. In the latter, the 3T3 feeder layer maintainsstemness of the cells.

FIG. 8 (40×magnification) depicts obvious colony formation of umbilicalcord mesenchymal cells (UCMC) isolated according to the inventioncultured on a 3T3 feeder layer at days 3 and 7(FIG. 8-1). The 3T3 feederlayer normally suppresses the growth of differentiated mesenchymal cellsas human dermal fibroblasts. Once again, this indicates a difference inbehavior of these mesenchymal cells as compared to their moredifferentiated counterparts. FIG. 8-2 shows the colony formingefficiency assay of the umbilical cord mesenchymal cells.

FIG. 9-1 to FIG. 9-28 show Western blot analysis by which the expressionof several embryonic stem cell markers in UCEC and UCMC isolatedaccording to the invention, was compared to the expression of thesemarkers in human dermal fibroblasts (NF), in bone marrow mesenchymalcells (BMSC) and adipose-derived mesenchymal cells (ADMC). FIGS. 9-29and 9-30 show secretion of Leukemia inhibitory factor detected byWestern blot analysis (FIG. 9-29) and highly secreted ActivinA andFollistatin detected by ELISA assay (FIG. 9-30), respectively insupernatants of umbilical cord mesenchymal and epithelial stem cellculture in comparison with bone marrow, adipose derived stem cells,human dermal fibroblasts and epidermal keratinocytes.

FIG. 10 shows indirect immunofluorescent analysis of markers ofepithelial cells expressed in umbilical cord epithelial stem cells suchas cytokeratins (CK)-general, CK17, CK6, CK10, CK19, CK18, CK16, CK15(FIG. 10-1); Hemidesmosome components-integrin alpha6, integrin beta4;Desmosome components (FIG. 10-2); Basement membrane components-laminin1,laminin5, collagen IV, collagen VII (FIG. 10-3) and other importantextracellular matrix components as integrin-betal and fibronectin (FIG.10-4).

FIG. 11-1 to FIG. 11-4 shows cytokine array analysis of secretedcytokines and growth factors by umbilical cord mesenchymal stem cells(UCMC) in comparison with human bone-marrow mesenchymal stem cells.

FIG. 12-1 to FIG. 12-7 shows cytokine array analysis of secretedcytokines and growth factors by umbilical cord epithelial stem cells(UCEC) in comparison with human epidermal keratinocytes.

FIG. 13 shows UCMC cells cultured in DMEM supplemented with 10% fetalcalf serum (FCS) (FIG. 13-1), serum-free media PTT-1 (FIG. 13-2), inserum-free media PTT-2 (FIG. 13-3, FIG. 13-4) and in serum-free mediaPTT-3 (FIG. 13-5). FIG. 13 also shows the growth of adipose derivedstromal cells (FIG. 13-6) and bone marrow derived stromal cells (FIG.13-7) in serum free medium PTT-3.

FIG. 14 shows global gene expression in umbilical cord epithelial andmesenchymal stem cells analyzed by DNA microarray. UCEC expressed atotal of 28055 genes and UCMC expressed a total of 34407 genes. Thereare 27308 overlapping genes expressing in both cell types. 747 genesexpressed were unique to UCEC and 7099 genes expressed were unique toUCMC. The selected genes of interest are presented in this Figure. Bothstem cell types expressed 140 genes related to embryonic stem cells andembryonic development.

FIG. 15 shows a schematic illustration of expansion of umbilical cordepithelial and mesenchymal stem cells using repetitive explants ofumbilical cord lining membrane tissues.

FIG. 16 depicts a cross section of an umbilical cord demonstrating theumbilical cord amniotic lining membrane (LM), the contained Wharton'sjelly (WJ), as well as two umbilical arteries (UA) and one umbilicalvein (UV) supported within this jelly.

FIG. 17 depicts direct (in-vitro) differentiation of epithelial cellsisolated from the amniotic membrane of umbilical cord (UCEC) into skinepidermal keratinocytes (FIG. 17A), and in-vitro differentiation ofmesenchymal cells isolated from the amniotic membrane of umbilical cord(UCMC) into osteoblasts (FIG. 17B).

DETAILED DESCRIPTION

The invention is based on the surprising finding that the amnioticmembrane of umbilical cord represents a source, from whichstem/progenitor cells such as mesenchymal and epithelial stem/progenitorcells can be successfully isolated and expanded under in vitroconditions. Even more surprising is the finding that these cells showembryonic stem cell-like characteristics. The amniotic membrane (alsocalled amniotic lining membrane), i.e. thin innermost membranous sacenclosing the placenta and developing embryo of mammals, has recentlybeen used as a natural substrate in ocular surface reconstruction and asa biological substrate for expanding limbal epithelial stem cells (cf.,e.g., Anderson, D. F. et al. (2001) Br. J. Ophthalmol. 85, 567-575;Grüterich, M. et al. (2003) Surv. Ophthalmol. 48, 631-646). However, nomethods have been described thus far for the isolation ofstem/progenitor cells from the amniotic membrane, at least for humans,nor has the amniotic membrane covering the umbilical cord been reportedas a source for stem cells.

The invention provides a method for isolating stem/progenitor cells fromthe amniotic membrane of umbilical cord, the method comprising:

-   -   (a) separating the amniotic membrane from the other components        of the umbilical cord in vitro;    -   (b) culturing the amniotic membrane tissue obtained in step (a)        under conditions allowing cell proliferation; and    -   (c) isolating the stem/progenitor cells.

For isolation of the cells of the invention from umbilical cord, theumbilical cord or a part thereof is usually collected immediately afterbirth (of a child in the case of humans) and for transport to thelaboratory transferred in a medium that is suitable for handling ofmammalian tissue. Examples of such media include, but are not limited toLeibovitz media which are commercially available from suppliers such asSigma Aldrich, Saint Louis, USA or HyClone, Logan, Utah, USA. Theumbilical cord is then typically processed under sterile conditions.Processing of the cord typically includes removing the blood that hasremained on the surface or within the blood vessels of the umbilicalcord by washing with a suitable buffer such as phosphate bufferedsaline. The umbilical cord is then typically reduced to smaller pieces,for example by cutting, and washed again before separating the amnioticmembrane from the other components. In this conjunction, it is notedthat it is not necessary to process the umbilical cord of a mammaliandonor immediately after birth but it is also possible, to collect theumbilical cord and, optionally after washing under sterile conditionsand reducing it into smaller pieces, to preserve the umbilical cord orparts thereof by cryo-preservation and to store the so obtainedspecimen, for example in liquid nitrogen, for later isolation of thecells of the invention from the umbilical cord. Accordingly, an (intact)umbilical cord or a portion of an intact umbilical cord that is treatedby cryo-preservation is also encompassed in the present invention. Inaddition, the umbilical cord amniotic membrane that has been separatedfrom the other components of the umbilical cord and is then treated bycryo-preservation is also encompassed in the present invention.

The term “cryo-preservation” is used herein in its regular meaning todescribe a process where cells or whole tissues are preserved by coolingto low sub-zero temperatures, such as (typically) −80° C. or −196° C.(the boiling point of liquid nitrogen). Cryo-preservation can be carriedout as known to the person skilled in the art and can include the use ofcryo-protectors such as dimethylsulfoxide (DMSO) or glycerol, which slowdown the formation of ice-crystals in the cells of the umbilical cord.

The term “stem/progenitor cell” as used herein refers to any cellderived of umbilical cord having the capacities to self-renewindefinitely and to differentiate in multiple cell or tissue types suchas endothelial cells, epithelial cells, fibroblasts, myocytes orneurons. Furthermore, the cells may be derived of any mammalian species,such as mouse, rat, guinea pig, rabbit, goat, dog, cat, sheep, monkey orhuman, with cells of human origin being preferred in one embodiment.

The term “embryonic stem cell-like properties” refers to the ability ofthe cells derived of umbilical cord that they can—almost like or exactlylike embryonic stem cells—differentiate spontaneously into all tissuetypes, meaning that they are pluripotent stem cells.

The term “amniotic membrane” as used herein refers to the thin innermostmembranous sac enclosing the developing embryo of mammals. Duringpregnancy, the fetus is surrounded and cushioned by a liquid calledamniotic fluid. This fluid, along with the fetus and the placenta, isenclosed within a sac called the amniotic membrane, which also coversthe umbilical cord. The amniotic fluid is important for several reasons.It cushions and protects the fetus, allowing the fetus to move freely.The amniotic fluid also allows the umbilical cord to float, preventingit from being compressed and cutting off the fetus' supply of oxygen andnutrients derived from the circulating blood within the placental bloodvessels. The amniotic sac contains the amniotic fluid which maintains ahomeostatic environment protecting the fetal environment from theoutside world. This barrier additionally protects the fetus fromorganisms (like bacteria or viruses) that could travel up the vagina andpotentially cause infection.

Media and reagents for tissue culture are well known in the art (cf.,for example, Pollard, J. W. and Walker, J. M. (1997) Basic Cell CultureProtocols, Second Edition, Humana Press, Totowa, N.J.; Freshney, R. I.(2000) Culture of Animal Cells, Fourth Edition, Wiley-Liss, Hoboken,N.J.). Examples of suitable media for incubating/transporting umbilicalcord tissue samples include, but are not limited to, Dulbecco's ModifiedEagle Medium (DMEM), RPMI media, Hanks' Balanced Salt Solution (HBSS)phosphate buffered saline (PBS), and L-15 medium, with the latter onebeing preferred in some embodiments. Examples of appropriate media forculturing stem/progenitor cells according to the invention include, butare not limited to, Dulbecco's Modified Eagle Medium (DMEM), DMEM-F12,RPMI media, EpiLlfe medium, and Medium 171, with the latter beingpreferred in some embodiments. The media may be supplemented with fetalcalf serum (FCS) or fetal bovine serum (FBS) as well as antibiotics,growth factors, amino acids, inhibitors or the like, which is wellwithin the general knowledge of the skilled artisan.

In one embodiment, the invention provides a method, further comprising:

-   -   (a″) separating these stem/progenitor cells from the amniotic        membrane tissue by a enzymatic digestion and/or direct tissue        explant technique before cultivation. The term “enzymatic        digestion technique” as used herein means that enzymes are added        to cleave the cells from the main tissue mass (here the amniotic        membrane of the umbilical cord). The separated cells are        subsequently collected. The term “direct tissue explant        technique” as used herein means that the tissue is first placed        in media without enzymes. Then under careful conditions the        cells separate from the main tissue mass by itself—and the cells        are then harvested for collection.

Methods for separating cells of a particular tissue or organ bytreatment with enzymes or by direct tissue explant are well known in theart (cf., for example, Pollard, J. W. and Walker, J. M. (1997) BasicCell Culture Protocols, Second Edition, Humana Press, Totowa, N.J.;Freshney, R. I. (2000) Culture of Animal Cells, Fourth Edition,Wiley-Liss, Hoboken, N.J.). Any enzyme catalyzing tissue dissociationmay be used for performing the methods of the present invention. In someembodiments, collagenase is used for that purpose. The enzyme may beused as a crude preparation or in purified form. It may be purified fromany prokaryotic or eukaryotic organism (with Clostridium histolyticumbeing most preferred) or produced recombinantly by means of genetechnology. Any type of collagenase may be employed, i.e. type 1, type2, type 3, type 4, or any combination thereof. In some embodiments theuse of collagenase type 1 is being preferred.

In one embodiment, the invention provides a method for isolatingstem/progenitor cells that have embryonic stem cell-like properties.These cells can ultimately be differentiated into, but not limited to,by morphology, epithelial or mesenchymal cells.

Accordingly, in another embodiment, the invention provides a method forisolating epithelial and/or mesenchymal stem/progenitor cells, whereinin accordance with the above disclosure these cells may have embryonicstem cell-like properties.

Epithelial stem/progenitor cells include any cells exhibiting aepithelial cell like morphology (i.e. a polyhedral shape) that can bedifferentiated into any type of epithelial cell such as, but not limitedto, skin epithelial cells, hair follicular cells, cornea epithelialcells, conjunctival epithelial cells, retinal epithelial cells, liverepithelial cells, kidney epithelial cells, pancreatic epithelial cells,oesophageal epithelial cells, small intestinal epithelial cells, largeintestinal epithelial cells, lung and airway epithelial cells, bladderepithelial cells or uterine epithelial cells.

Mesenchymal stem/progenitor cells include any cells exhibiting amesenchymal cell like morphology (i.e. a spindle-like shape) that can bedifferentiated into any type of mesenchymal cell such as, but notlimited to, skin fibroblasts, chondrocytes, osteoblasts, tenocytes,ligament fibroblasts, cardiomyocytes, smooth muscle cells, skeletalmuscle cells, adipocytes, cells derived from endocrine glands, and allvarieties and derivatives of neurectodermal cells.

In another embodiment, the invention provides a method furthercomprising:

-   -   (d) culturing the stem/progenitor cells under conditions        allowing the cells to undergo clonal expansion.

The term “clonal expansion” (sometimes also referred to as “mitoticclonal expansion”) relates to a process that occurs early in thedifferentiation program of a cell, by which stem/progenitor cells becomecommitted to a particular lineage and then undergo terminaldifferentiation. It is well known in the art that the conditions toinduce clonal expansion of progenitor cells may vary significantlybetween different cell types. Without being limited to a particularmethod, the induction of clonal expansion is generally achieved bycultivating the stem/progenitor cells in a medium that has beenoptimized for cell proliferation. Such media are commercially availablefrom many providers. Non-limiting examples of such media areKGM®-Keratinocyte Medium (Cambrex), MEGM-Mammary Epithelial Cell Medium(Cambrex), EpiLife medium (Cascade Biologics) or Medium 171 (CascadeBiologics). Alternatively, a culture medium may be supplemented withreagents inducing cell proliferation such as growth factors. Suchreagents may be admixed in a single solution such as the HumanKeratinocyte Growth Supplement Kit (Cascade Biologics), to name oneexample, or may be supplemented individually. Such reagents include, butare not limited to, growth factors (such as epidermal growth factor,insulin-like growth factor-1, platelet-derived growth factor-BB,transforming growth factor-β1, insulin, for example), hormones (such asa bovine pituitary extract), hydrocortisone, transferrin and the like inany suitable combination to induce clonal expansion of a given celltype. The term “clonal expansion” also includes cultivation of the cellin vivo, for example, by injection of the cells into mammals such ashumans, mice, rats, monkeys, apes to name only a few.

In yet another embodiment, the invention provides a method furthercomprising:

-   -   (e) culturing the stem/progenitor cells under conditions        allowing the differentiation of said cells into epithelial cells        and/or mesenchymal cells; and    -   (f) isolating the differentiated cells.        Thus, the invention also provides for a method of        differentiating a stem/progenitor cell into a differentiated        cell.

In yet another embodiment, the invention provides a method, furthercomprising:

-   -   (g) preserving the isolated stem/progenitor cells for further        use.

Methods and protocols for preserving and storing of eukaryotic cells,and in particular mammalian cells, are well known in the art (cf., forexample, Pollard, J. W. and Walker, J. M. (1997) Basic Cell CultureProtocols, Second Edition, Humana Press, Totowa, N.J.; Freshney, R. I.(2000) Culture of Animal Cells, Fourth Edition, Wiley-Liss, Hoboken,N.J.). Any method maintaining the biological activity of the isolatedstem/progenitor cells such as epithelial or mesenchymal stem/progenitorcells may be utilized in connection with the present invention. In onepreferred embodiment, the stem/progenitor cells are maintained andstored by using cryo-preservation.

Accordingly, the invention is also directed to a progenitor/stem cellderived from the amniotic membrane of umbilical cord by means of theabove methods and to a cell differentiated from the progenitor/stemcell. In addition, the invention is also directed to a cell bankcomprising or consisting of one or more progenitor/stem cells that havebeen isolated as described here. This cell bank of progenitor/stem cellsmay be autologous to an individual or pooled (the latter for subsequentallogeneic transplantation, for example), and subsequently can beemployed by further differentiation for regenerative medicine, tissuerepair and regeneration, for example.

In accordance with the above, the invention is also directed to apharmaceutical composition comprising a stem/progenitor cell isolatedfrom the amniotic membrane of umbilical cord by the above inventivemethod. The pharmaceutical composition can also include a celldifferentiated from the stem/progenitor cell. The pharmaceuticalcomposition can be of any kind, and usually comprises thestem/progenitor cells, a cell differentiated therefrom or a cellularsecretion or cellular extract thereof together with a suitabletherapeutically acceptable carrier/excipient. In case of a cellularsecretion, the desired compound(s) can be used in some embodiments inthe form of the supernatant into which the compound(s) is/are secreted.In other embodiment, the supernatant might be processed, for example, bypurification and concentration prior to be included in a pharmaceuticalcomposition. In some embodiments, the pharmaceutical composition isadapted for systemic or topical application.

A pharmaceutical composition adapted for topical application may be inliquid or viscous form. Examples thereof include an ointment, a cream,and a lotion and the like. Examples for pharmaceutical compositions thatare suitable for systemic use are liquid compositions, wherein thestem/progenitor cells or the cellular extract are dissolved in a bufferthat is acceptable for injection or infusion, for example. Thepreparation of such pharmaceutical compositions is within the knowledgeof the person skilled in the art and described in Gennaro, A. L. andGennaro, A. R. (2000) Remington: The Science and Practice of Pharmacy,20th Ed., Lippincott Williams & Wilkins, Philadelphia, Pa., for example.

Accordingly, the invention also relates to a method of treating asubject having a disorder. This method comprises administering to thesubject an effective amount either of a stem/progenitor cell isolated asexplained herein or of a cellular extract derived from such a cell.

In principle, any condition or disorder which is suitable for beingtreated by means of stem cells/progenitor cells can be treated with acell or a cellular extract of present invention. It is also possible todifferentiate cells of the invention into a desired type of cell, forexample, but not limited to, a skin cell, a bone cell, an hormoneproducing cell such as a beta islet insulin producing cell, and use thedifferentiated cell therapeutically. In some embodiments, the disorderis selected from the group consisting of neoplastic disease, acceleratedskin aging and skin disorders, tissue disorders, visceral endocrinedeficiencies, and neural disorders.

The tissue disorder to be treated can be a congenital or an acquiredtissue deficiency. Examples of visceral endocrine deficiency that can betreated with a cell of the invention include, but are not limited to,Diabetes mellitus associated with insulin deficiency, testosteronedeficiency, anemia, hypoglycemia, hyperglycemia, pancreatic deficiency,adrenal deficiency, and thyroid deficiencies.

Examples of neural disorders that can be treated include, but are notlimited to, Alzheimer's disease, Parkinson's disease, Jacob Kreutzfeld'sdisease, Lou Gehrig's disease, Huntington's disease and neuralneoplastic conditions.

An example of a skin disease is a wound or a damaged part of the skin,for example, sun burned skin. Also aging of the skin is considered to bea skin disease herein. Topical or similar delivery of stem/progenitorcells of the invention or cellular extracts thereof, for example, as aconstituent in lotions or creams or any other suitable vehicle may thusbe used for repair of sun damaged skin and in addition may slow alsodown the aging process of skin (anti-aging properties) by replenishing,and thus fortifying, deficient growth factors and related peptideelements, without which skin aging would be accelerated. Thestem/progenitor cells may also migrate to injured regions of the bodysuch as surface wounds to form the necessary required cellular elementsnecessary for the local reparative processes (cf. The Journal ofImmunology, 2001, 166: 7556-7562; or International Journal ofBiochemical and Cell Biology 2004; 36: 598-606.

The neoplastic disease may be cancer, in particular as recent studieshave demonstrated that stem cells may selectively target neoplastictumor tissue (Journal of the National Cancer Institute 2004; 96 (21):1593-1603) allowing for directed delivery of antineoplastic agents suchas interferon to neoplastic foci. The cancer can be any kind of cancer,including those cancers that are able to form solid tumors, ranging fromskin cancer to cancer of the internal organs. Examples of cancers to betreaded include, squamous cell carcinoma, breast ductal and lobularcarcinoma, hepatocellular carcinoma, nasopharyngeal carcinoma, lungcancer, bone cancer, pancreatic cancer, skin cancer, cancer of the heador neck, cutaneous or intraocular malignant melanoma, uterine cancer,ovarian cancer, rectal cancer, cancer of the anal region, stomachcancer, colon cancer, breast cancer, testicular cancer, uterine cancer,carcinoma of the fallopian tubes, carcinoma of the endometrium,carcinoma of the cervix, carcinoma of the vagina, carcinoma of thevulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of theesophagus, cancer of the small intestine, cancer of the endocrinesystem, cancer of the thyroid gland, cancer of the parathyroid gland,cancer of the adrenal gland, sarcoma of soft tissue, cancer of theurethra, cancer of the penis, prostate cancer, chronic or acuteleukemias, solid tumors of childhood, lymphocytic lymphoma, cancer ofthe bladder, cancer of the kidney or ureter, renal cell carcinoma,carcinoma of the renal pelvis, neoplasm of the central nervous system(CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor,brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoidcancer or any combination of such cancers, including disseminated(metastasising) forms thereof. In case of treatment of a neoplasticdisease the umbilical cord amnion derived stem cells and/or theircellular extracts disclosed herein can be administered systemically bothas a direct treatment and/or as a carrier vehicle. In the latter case ofanti-neoplastic tumor therapy, the cells comprise an anti-neoplasticagent.

In another pharmaceutical use, stem/progenitor cells of the presentinvention can be used for gene therapy. For this purpose, the cells canbe transformed with a nucleic acid encoding the protein that is to beproduced in the cells. The nucleic acid can be introduced into a cellsof the invention using any of the various methods that are well known tothe skilled person, for example, using a viral vector and/or a lipidcontaining transfection composition such as as IBAfect (IBA GmbH,Göttingen, Germany), Fugene (Roche), GenePorter (Gene Therapy Systems),Lipofectamine (Invitrogen), Superfect (Qiagen), Metafecten (Biontex) orthose ones described in the PCT application WO 01/015755). In a relatedembodiment, the cells of the invention, after being transformed with anucleic acid encoding a polypeptide of choice, can be used ofrecombinantly producing this polypeptide.

As mentioned above, stem cell extracts are rich in a variety of growthfactors and peptides that are relevant for normal tissue physiology.Such growth factors and/or peptides may be deficient in exposed parts ofthe body, such as the skin, which is the surface layer of all humanbeings protecting the body from external elements for the maintenance ofinternal homeostasis. Therefore in a further embodiment, stem/progenitorcells of the invention or cellular extracts thereof are suitable for thetreatment and/or maintenance of internal homeostasis.

In a further embodiment and in line with the above disclosure, thestem/progenitor cells of the invention can be used for the production ofany biological molecule. The biological molecule can be, for instance,any molecule that is naturally produced in the cells or a molecule thecoding nucleic acid of which has been introduced into the cells viarecombinant DNA technology. Examples of molecules that can be producedby the cells of the invention include, but are not limited to, a proteinsuch as a cytokine, a growth factor such as insulin-like growth factor(IGF), epidermal growth factor (EGF), transforming growth factor beta(TGF-beta), Activin A, a bone morphogenetic protein (BMP), PDGF or ahormone as insulin or erythropoietin or a transporter protein suchtransferrin, a peptide such a growth factor or hormone (e.g. luteinichormone (LSH), follicle stimulating hormone (FSH)), a small organicmolecule such as a steroid hormone, an oligo- or polysaccharide, forexample, heparin or heparan sulfate (cf., example WO 96/23003, or WO96/02259 in this regard), a proteoglycan, a glycoprotein such ascollagen or laminin, or a lipid, to name only a few.

In a further aspect and in accordance with recent approaches (see, forexample, Amit, M et al., Human feeder layers for human embryonic stellcells, Biol Reprod 2003; 68: 2150-2156), the stem/progenitor cellsdescribed here can be used as feeder layer for the cultivation of otherembryonic stem cells, in particular human embryonic stem cells. In oneof these embodiments the cells of the present invention are preferablyof human origin, since using human cells as feeder layer minimizes therisk of contaminating the cell culture with animal-derived componentssuch as animal pathogens or immunogens. In this respect, it is to benoted that the cells of the invention can be cultivated under serum freeconditions. Accordingly, employing the cells as feeder layer andcultivating the cell culture under with serum free media as the onedescribed herein later, or in Draper et al. (Culture andcharacterization of human embryonic stem cell lines, Stem Cells Dev2004, 13:325-336) or in the International patent application WO98/30679, for example.

In this connection, it is noted that in transplantation surgery andcell-based therapy high quantities of low passage cells with a minimalproportion of senescent cells (i.e., large proportion of high qualitycells) are crucial and are required to be derived within the shortestpossible time during cell expansion. For example, mesenchymal stem cellsfrom bone marrow and cord blood are low in quantity and thereforerequire expansion over many passages for a long period of time in orderto achieve the sufficient number of cells required for cell transplant.The high passage cells however tend to deteriorate in quality and maylead to cell senescence or cancerous transformation. It has been foundhere that high quantities of cells of the present invention can beobtained by low passage numbers using a repetitive explantationtechnique. The present invention thus also relates to a method ofcultivating stem/progenitors cells of the invention, wherein this methodcomprises:

-   -   Obtaining a tissue explant from the amniotic membrane of        umbilical cord;    -   Cultivating the tissue explant in suitable cultivation media and        cultivation conditions over a suitable period of time,    -   Optionally exposing the tissue explant to fresh cultivation        media and continuing the cultivation under suitable conditions        over a suitable period of time (cf., FIG. 15).

The cultivation can be carried out in for as many cycles (passages) aswanted and be stopped once the desired number of cells has beenobtained. Exposing the tissue explant to fresh cultivation can becarried out by removing the used cell cultivation medium from the vesselused for growing the cells and adding fresh media to that vessel.Instead of replacing the media in the used vessel, exposing to freshcultivation media can be achieved by transferring the tissue explant toa new vessel which is filled with cultivation media. The tissue explantused for cultivation/propagation of the cells can be obtained by anysuitable method, for example by the “direct tissue explant technique” asexplained above (in which the tissue is first placed in media withoutenzymes, and then under careful conditions the cells separate from themain tissue mass by itself—and the cells are then harvested forcollection).

The cultivation of the tissue explants can be carried out in any mediathat is suitable for cultivation of mammalian cells. Examples includethe conventional and commercially available media that are given abovewith respect to the cultivation or the clonal expansion of the cells ofthe invention such as, but not limited to, KGM®-Keratinocyte Medium(Cambrex), MEGM—Mammary Epithelial Cell Medium (Cambrex) EpiLife medium(Cascade Biologics), Medium 171 (Cascade Biologics), DMEM, DMEM-F12 orRPMI media. The cultivation is typically carried out at conditions(temperature, atmosphere) that are normally used for cultivation ofcells of the species of which the cells are derived, for example, at 37°C. in air atmosphere with 5% CO₂. In one embodiment, the cultivation iscarried out using serum free, in particular bovine serum free media. Thecultivation (in one passage) is performed for any suitable time thecells need for growth, typically, but by no means limited to, for about1 to several days, for example to about 7 or about 8 days.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including”, “containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the inventions embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims and non-limitingexamples. In addition, where features or aspects of the invention aredescribed in terms of Markush groups, those skilled in the art willrecognize that the invention is also thereby described in terms of anyindividual member or subgroup of members of the Markush group.

EXAMPLES Example 1 Collection of Umbilical Cord Tissue

Umbilical cord tissue is collected immediately after delivery of thechild. The specimen is rinsed clean and immediately transferred into a500 ml sterile glass bottle containing culture transport medium (L-15medium supplemented with 50 IU/ml penicillin, 50 μg/ml streptomycin, 250μg/ml fungizone, 50 μg/ml gentamicin; all reagents purchased fromInvitrogen) prior to transport to the laboratory. In the laboratory,stem cell extraction is conducted in a laminar flow hood under sterileconditions. The specimen is first transferred to a sterile stainlesssteel tray. All remaining blood in the cord vessels is removed bymultiple syringing washes using warm phosphate-buffered saline (PBS)supplemented with 5 IU/ml heparin (from Sigma). Plain PBS withoutheparin is used in the final washes. The umbilical cord tissue specimenis then cut into pieces 2 cm in length and transferred into 10 cmdiameter cell culture dishes, where further washing and disinfection isperformed with 70% ethanol followed by multiple washes using PBScontaining an antibiotic mixture (50 IU/ml penicillin, 50 μg/mlstreptomycin, 250 μg/ml fungizone, 50 μg/ml gentamicin; all purchasedfrom Invitrogen) until the solution becomes clear.

Example 2 Cell Separation/Cultivation

Dissection of umbilical cord tissue is first performed to separate theumbilical cord amniotic membrane from Wharton's jelly (i.e. the matrixof umbilical cord) and other internal components. The isolated amnioticmembrane is then cut into small pieces (0.5 cm×0.5 cm) for cellisolation. Explant is performed by placing the pieces of umbilical cordamniotic membrane on tissue culture dishes at different cell cultureconditions for isolation of either epithelial or mesenchymal stem cells.

For mesenchymal cell separation/cultivation, the explants were submergedin 5 ml DMEM (Invitrogen) supplemented with 10% fetal bovine serum(Hyclone) (DMEM/10% FBS) and maintained in a CO₂ cell culture incubatorat 37° C. The medium was changed every 2 or 3 days. Cell outgrowth wasmonitored under light microscopy. Outgrowing cells were harvested bytrypsinization (0.125% trypsin/0.05% EDTA) for further expansion andcryo-preservation using DMEM/10% FBS.

For epithelial cell separation/cultivation, cell culture plasticsurfaces were coated with collagen 1/collagen 4 mixtures (1:2) beforeplacing the tissue samples on the surface. The tissue samples weresubmerged in 5 ml EpiLife medium or Medium 171 (both from CascadeBiologics). The medium was changed every 2 or 3 days. Cell outgrowthfrom tissue culture explants was monitored under light microscopy.Outgrowing cells were harvested by trypsinization (0.125% trypsin/0.05%EDTA) using EpiLife medium or Medium 171.

For the enzymatic extraction method of cells, umbilical cord amnioticmembrane was divided into small pieces of 0.5 cm×0.5 cm and digested in0.1% (w/v) collagenase typel solution (Roche Diagnostics) at 37° C. for6 hours. The samples were vortexed every 15 min for 2 min. Cells wereharvested by centrifugation at 4000 rpm for 30 min. Two differentapproaches were employed to isolate either epithelial or mesenchymalstem cells.

For isolation of epithelial stem cells, cell pellets were resuspended inEpiLife medium or Medium 171 (both from Cascade Biologics) supplementedwith 50 μg/ml insulin-like growth factor-1 (IGF-1), 50 μg/mlplatelet-derived growth factor-BB (PDGF-BB), 5 μg/ml transforming growthfactor-β1 (TGF-β1), and 5 μg/ml insulin (all obtained from R&D Systems),counted and seeded on 10 cm tissue culture dishes pre-coated withcollagen 1/collagen 4 mixtures (1:2; Becton Dickinson) at density of1×10⁶ cells/dish. After 24 hours, attached cells were washed with warmPBS and medium was replaced with supplement-added EpiLife medium orMedium 171. The medium was changed every 2 or 3 days. Cell growth andexpanding clonal formation was monitored under light microscopy. At aconfluence of about 70%, cells were sub-cultured by trypsinization(0.125% trypsin/0.05% EDTA) for further expansion and cryo-preservation.

For isolation of mesenchymal stem cells, cell pellets were resuspendedin DMEM/10% FBS, counted and seeded on 10 cm tissue culture dishes atdensity of 1×10⁶ cells/dish. The culture medium was changed every 2 or 3days. Cell growth and expansion was monitored under light microscopy. Ata confluence of about 90%, cells were sub-cultured as outlined above.

For cultivation of epithelial and mesenchymal stem cells on feederlayer, umbilical cord lining membrane was digested by collagenasetreatment, counted and seeded on 10 cm tissue culture dishes coated withlethally irradiated or Mitomycin C treated 3T3 fibroblasts (feederlayer) in Green's medium. The culture medium was changed every 2 or 3days. Colony formation was monitored under light microscopy andphotographed.

Example 3 Identification of Stem/Progenitor Cells

Epithelial cells: FIG. 1 shows pictures of outgrowing epithelial cellsfrom umbilical cord amniotic membrane prepared by the method usingtissue explant (40×magnification). Pictures were taken at day 2 (FIG.1A) and day 5 (FIG. 1B, C) of tissue culture. Cell morphology analysisdemonstrated polyhedral shaped epithelial-like cells. Enzymaticdigestion of the umbilical cord segments yielded similar (FIG. 2),epithelial cells at day 2 (FIG. A, C) and day 5 (FIG. B, D)(40×magnification). FIG. 7 shows pictures of colony formation ofepithelial stem cells from umbilical cord amniotic membrane cultured onfeeder layer using Green's method (40×magnification). A colony ofpolyhedral shaped epithelial-like cells expanded rapidly from day 3 today 7.

Mesenchymal cells: Outgrowth of mesenchymal cells explanted fromumbilical cord amniotic membrane was observed as early as 48 hours afterplacement in tissue culture dishes using DMEM supplemented with 10%fetal calf serum (FCS) as culture medium (FIG. 3A, C)(40×magnification). The cells were characterized by their spindle shapedmorphology, and migrated and expanded both easily and quickly in vitro,closely resembling fibroblasts (FIG. 3B, D) (40×magnification). Similarobservations were noted in the cell group isolated by collagenaseenzymatic digestion (FIG. 4). FIG. 4A shows mesenchymal cells isolatedfrom umbilical cord amniotic membrane at day 2. Cell proliferation wasobserved at day 5 (FIG. 4B) (40×magnification). FIGS. 6 and 8-1 showpictures of colony formation of mesenchymal stem cells from umbilicalcord amniotic membrane cultured on non-feeder layer (FIG. 6) and feederlayer condition (FIG. 8-1, using a 3T3 feeder layer) in DMEM/10% FCS (40x magnification). The colonies of elongated shaped fibroblastic-likecells expanded rapidly from day 3 to day 7. It is noted in this respect,that the 3T3 feeder layer normally suppresses the growth of mesenchymalcells as human dermal fibroblasts. Once again, this indicates adifference in the behavior of the mesenchymal cells of the invention ascompared to more differentiated counterparts.

In further experiments the colony forming ability of the mesenchymalcells of the invention (UCMC) was studied. For colony forming efficiencyassay, 100-200 single cells were seeded in 100 mm tissue culture dishesor T75 flasks without feeder layers. Cells were maintained in DMEM/10%FCS for 12 days. Single colony formation was monitored under theinverted light microscope (experiment was carried out in duplicate,experiments termed UCMC-16 and UCMC-17 in FIG. 8-2). Microphotographswere sequentially taken. At day 12, colonies were fixed and stained withRhodamine. UCMC colony forming units were seen (FIG. 8-2). The multiplelarge colonies observed, indicated self-renewal of UMCM in-vitro (FIG.8-2).

Western blot analysis (FIG. 9) shows that mesenchymal stem cells fromumbilical cord amniotic membrane (UCMC) and umbilical cord epithelialcells (UCEC) isolated in accordance with the invention expressed thePOU5f1 gene which encodes the transcription factor Octamer-4 (Oct-4) aspecific marker of embryonic stem cells (cf. Niwa, H., Miyazaki, J., andSmith, A. G. (2000). Nat. Genet. 24, 372-376) (FIG. 9-1). Thus, thisanalysis indicates the embryonic-like properties of these stem cells.These mesenchymal and epithelial cells also expressed Bmi-1, a markerthat is required for the self-renewal of adult stem cells (cf., Park etal., J. Clin. Invest. 113,175-179 (2004) (FIG. 9-27) as well as leukemiainhibitory factor (LIF) (FIG. 9-28) that is considered to maintain thepluripotency of stem cells and embryonic cells and has thus, for examplebeen used for isolation and expansion of human neural stem cells. Thesecells also highly expressed the other growth factors such as connectivetissue growth factor (CTGF) (FIGS. 9-6, 9-7), vascular endothelialgrowth factor (VEGF) (FIGS. 9-10, 9-11), placenta-like growth factorPLGF (FIGS. 9-4, 9-5), STAT3 (FIGS. 9-2, 9-3), stem cell factor (SCF)(FIG. 9-16), Hepatoma-derived Growth Factor (HDGF) (FIGS. 9-14, 9-15),Fibroblast Growth Factor-2 (FGF-2) (FIGS. 9-12, 9-13), Platelet-derivedGrowth Factor (PDGF) (FIGS. 9-8, 9-9), alpha-Smooth Muscle Actin (α-SMA)(FIG. 9-17), Fibronectin (FIGS. 9-18, 9-19), Decorin (FIG. 9-20),Syndecan-1,2,3,4 (FIGS. 9-21 to 9-26). In FIG. 9, the expression ofthese genes is compared to human dermal fibroblasts, bone marrowmesenchymal cells (BMSC) and adipose-derived mesenchymal cells (ADMC).FIG. 9-29 shows Western blot data of the secretion of leukemiainhibitory factor (LIF) by both UCEC and UCMC. FIG. 9-30 shows highlysecreted Activin A and Follistatin (both of which proteins are wellknown to promote tissue repair and regeneration, enhanced angiogenesis,and maintain embryonic stem cell culture, so that expression of therespective genes is a sign for the embryonic properties and ability ofthe cells to differentiate) detected ELISA assay (FIG. 9-30) insupernatants of umbilical cord mesenchymal and epithelial stem cellculture in comparison with bone marrow, adipose derived stem cells,human dermal fibroblasts and epidermal keratinocytes. Also these resultsindicate that the cells of the invention are promising candidates intherapeutic application of these cells areas such as regenerativemedicine, aging medicine, tissue repair and tissue engineering. Inaddition, FIGS. 9-29 and 9-30 show the capability of the cells to secretan expression product into the culture medium.

Mesenchymal cells were further characterized by analysis of secretedcytokines and growth factors in comparison with human bone-marrowmesenchymal stem cells. The umbilical cord epithelial stem cells (UCEC)were analysed in comparison with human epidermal keratinocytes. Thisanalysis was carried out as follows: Briefly, UCMC, UCEC, dermalfibroblasts, bone-marrow mesenchymal cells, epidermal keratinocytes werecultured in growth media until 100% confluence (37° C., 5% CO₂) and thensynchronized in starvation medium (serum-free DMEM) for 48 hours. Thenext day, the medium was replaced the next against fresh serum-free DMEMand the cells then were cultivated for another 48 hours. Conditionedmedia were collected, concentrated and analyzed using a Cytokine Array(RayBiotech, Inc, GA, USA).

The results of this analysis show that UCMC secrete Interleukin-6(IL-6); (MCP1); hepatocyte growth factor (HGF); Interleukin-8 (IL8);sTNFR1; GRO; TIMP1; TIMP2; TRAILR3; uPAR; ICAM1; IGFBP3; IGFBP6 (FIG.11), whereas UCEC secrete IGFBP-4; PARC; EGF; IGFBP-2; IL-6; Angiogenin;GCP-2; IL1Rα; MCP-1; RANTES; SCF; TNFβ; HGF; 1L8; sTNFR; GRO; GRO-α;Amphiregulin; IL-1 R4/ST2; TIMP1; TIMP2; uPAR; VEGF (FIG. 12).

Accordingly, this shows that both cells types secrete large amounts ofcytokines and growth factors that play important roles in developmentalbiology, tissue homeostasis, tissue repair and regeneration andangiogenesis. This further demonstrates the versatility of the cells ofthe invention for use in the respective therapeutic applications.

In addition, the cells of the invention were further examined withrespect to their safety profile using mouse teratoma formation assay asan indicator. Six SCID mice were used in these experiments. A suspensionof more than 2 million UCMC was injected with a sterile 25G needle intothe thigh muscle of each SCID mouse. Animals were kept up to 6 monthsand tumor formation was assessed. No tumor formation was observed inthese mice (data not shown). This indicates that the cells of theinvention are safe and do not have any capability to form tumors, benignor otherwise.

The UCMC were also analysed for their expression of human leukocyteantigen (HLA) molecules. When testing on major histocompatibilitycomplex (MHC) class I molecules, this analysis showed that HLA-Amolecules were present in high number (test result in arbitrary unit:3201), meaning that the cells are HLA-A positive whereas expression ofHLA-B molecules was insignificant (test result in arbitrary units: 35),meaning the cells are HLA-B negative. As HLA-B is mainly responsible forrejection reaction in transplantation, this result indicates that thecells of the invention are not only suitable for autologoustransplantation but also for allogeneic transplantation. The cells weretested positive for Class II MHC molecule HLA-DR52 and tested negativefor Class II MHC molecule HLA-DRB4. HLA-DRB1 was also found to bepresent (0301/05/20/22,

Example 4 Cultivation of Stem/Progenitor Cells in Serum Free Media

UCMC cells were cultured in DMEM containing 10 FCS and in serum-freemedia, PTT-1, PTT-2 and PTT-3. The three media PTT-1, PTT-2 and PTT-3were prepared by one of the present inventors, Dr Phan. In brief, these3 media do not contain fetal bovine or human serum, but containdifferent cytokines and growth factors such as IGF, EGF, TGF-beta,Activin A, BMPs, PDGF, transferrin, and insulin. The growth factorcomponents vary between media to assess differential growthcharacteristics. The cultivation was carried out as follows: Differentproportions of growth factors and cytokines were added in basal media.UCMC were thawed and maintained in these media for 10 days. Cellproliferation was monitored under light microscopy.

FIG. 13 shows good UCMC growth in the 4 different media groups (FIG.13-1 to FIG. 13-5), wherein the morphology of UCMC cells is differentdepending on the ratio or proportion of cytokines or growth factorspresent in the respective media. In contrast, bone marrow andadipose-derived mesenchymal cells did not grow well in these serum-freemedia (FIG. 13-6 and FIG. 13-7). Accordingly, the good growth of theUCMC demonstrates the robustness of the cells of the invention and theirhigh viability, indicating that their growth characteristics aresuperior to conventional sources of mesenchymal stem cells as bonemarrow derived and adipose-derived mesenchymal cells. In this respect,it is worth to note that (bovine) serum free medium was used in theseexperiments and that the majority of human mesenchymal cells do not growwell in serum-free medium systems. Thus, using the cells of theinvention in connection with defined serum-free media technologies is abig advantage in cell therapy as the risks of using fetal bovine serumfor cell culture and expansion are removed. (Although use of bovineserum has been practiced for a long time and typically optimizes cellgrowth, concerns of its used have been raised as to the transmission ofzoonoses as Bovine Spongiform Encephalopathy (Mad Cow Disease)).

Example 5 Characterization of the Gene Expression Profile of UmbilicalCord Epithelial and Mesenchymal Stem Cells

The gene expression profile of umbilical cord epithelial and mesenchymalstem cells was analyzed using a DNA microarray. For this purpose, UCMCand UCEC were cultured in growth media at 37° C., 5% CO₂ until 100%confluence. Cells were synchronized in basal media for 48 hours thenreplaced with fresh basal media for another 48 hours. Total RNA washarvested and sent to Silicon Genetics Microarray Service. Data analysiswas performed using GeneSpring 7.2). FIG. 14 summarizes the global geneexpression. UCEC expressed a total of 28055 genes and UCMC expressed atotal of 34407 genes. There are 27308 overlapping genes expressing inboth cell types. 747 genes expressed were unique to UCEC and 7099 genesexpressed were unique to UCMC. The selected genes of interest arepresented in FIG. 14.

Both stem cell types expressed 140 genes related to embryonic stem cellsand embryonic development, further supporting that the cells of theinvention have embryonic stem cell-like properties: Nanog; Alpha-fetalprotein; Pre-B-cell leukemia transcription factor 3; Laminin alpha 5;Carcinoembryonic antigen-like 1; abhydrolase domain containing 2;Delta-like 3 (Drosophila); Muscleblind-like (Drosophila); GNAS complexlocus; Carcinoembryonic antigen-related cell adhesion molecule 3;Palmitoyl-protein thioesterase 2; Pregnancy specific beta-1-glycoprotein2; Carcinoembryonic antigen-like 1; Embryonic ectoderm development;Maternal embryonic leucine zipper kinase; Chorionic somatomammotropinhormone 2; Forkhead box D3; radical fringe homolog (Drosophila); Kinesinfamily member 1B; Myosin, heavy polypeptide 3, skeletal muscle,embryonic; Split hand/foot malformation (ectrodactyly) type 3; TEAdomain family member 3; Laminin, alpha 1; Chorionic somatomammotropinhormone 1; placental lactogen; Corticotropin releasing hormone receptor1; thyrotrophic embryonic factor; Aryl-hydrocarbon receptor nucleartranslocator 2; Membrane frizzled-related protein; Neuregulin1′Collagen, type XVI, alpha 1; Neuregulin 1; Chorionic somatomammotropinhormone 1 (placental lactogen); CUG triplet repeat, RNA binding protein1; Chorionic somatomammotropin hormone 1 (placental lactogen)Bystin-like; MyoD family inhibitor; Retinoic acid induced 2; GNAScomplex locus; Pre-B-cell leukemia transcription factor 4; Laminin,alpha 2 (merosin, congenital muscular dystrophy); SMAD, mothers againstDPP homolog 1 (Drosophila); Homo sapiens transcribed sequence withmoderate similarity to protein pir:D28928 (H. sapiens) D28928pregnancy-specific beta-1 glycoprotein IB, abortive—human (fragment);Kinesin family member 1B; Bruno-like 4, RNA binding protein(Drosophila); Embryo brain specific protein; Pregnancy-induced growthinhibitor; SMAD, mothers against DPP homolog 5 (Drosophila); Chorionicsomatomammotropin hormone 2;Adenylate cyclase activating polypeptide 1(pituitary); Carcinoembryonic antigen-related cell adhesion molecule;Laminin, alpha 3; Protein O-fucosyltransferase 1; Jagged 1 (Alagillesyndrome); Twisted gastrulation homolog 1 (Drosophila); ELAV (embryoniclethal, abnormal vision, Drosophila)-like 3 (Hu antigen C); Thyrotrophicembryonic factor; Solute carrier family 43, member 3; Inversin;nephronophthisis 2 (infantile); inversion of embryonic turning; Homosapiens inversin (INVS), transcript variant 2, mRNA; Homo sapienstranscribed sequences; Homeo box D8; Embryonal Fyn-associated substrate;ELAV (embryonic lethal, abnormal vision, Drosophila)-like 1 (Hu antigenR); Basic helix-loop-helix domain containing, class B, 2; Oxytocinreceptor; Teratocarcinoma-derived growth factor 1; Fms-related tyrosinekinase 1 (vascular endothelial growth factor/vascular permeabilityfactor receptor); Adrenomedullin; Nuclear receptor coactivator 6-CUGtriplet repeat, RNA binding protein 1; Twisted gastrulation homolog 1(Drosophila); Carcinoembryonic antigen-related cell adhesion molecule4;Protein tyrosine phosphatase, receptor type, R; Acrg embryoniclethality (mouse) minimal region ortholog; EPH receptor A3;Delta-like 1(Drosophila); Nasal embryonic LHRH factor; Transcription factor CP2-like1; Split hand/foot malformation (ectrodactyly) type 3; Jagged 2; Homosapiens transcribed sequence; Neuregulin 1; Split hand/foot malformation(ectrodactyly) type 1; Solute carrier family 43, member 3;Hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-Coenzyme Athiolase/enoyl-Coenzyme A hydratase (trifunctional protein), alphasubunit; Fucosyltransferase 10 (alpha (1,3) fucosyltransferase); Acrgembryonic lethality (mouse) minimal region ortholog; Carcinoembryonicantigen-related cell adhesion molecule 7; Nucleophosmin/nucleoplasmin,2; Fc fragment of IgG, receptor, transporter, alpha; Twistedgastrulation homolog 1 (Drosophila); Homo sapiens similar to vacuolarprotein sorting 35; maternal-embryonic 3 (LOC146485), mRNA; abhydrolasedomain containing 2; T, brachyury homolog (mouse); A disintegrin andmetalloproteinase domain 10; Ribosomal protein L29; Endothelinconverting enzyme 2; ELAV (embryonic lethal, abnormal vision,Drosophila)-like 1 (Hu antigen R); Trophinin; Homeo box B6; Laminin,alpha 4; Homeo box B6; hypothetical protein FLJ13456; NACHT, leucinerich repeat and PYD containing 5; ELAV (embryonic lethal, abnormalvision, Drosophila)-like 1 (Hu antigen R); Undifferentiated embryoniccell transcription factor 1; Pregnancy-associated plasma protein A,pappalysin 1; Secretoglobin, family 1A, member 1 (uteroglobin);Parathyroid hormone-like hormone; Carcinoembryonic antigen-related celladhesion molecule 1 (biliary glycoprotein); Laminin, alpha 1.

Both stem cell types also expressed thousands of genes related todevelopmental biology, cell growth and differentiation, cellhomeostasis, cell and tissue repair and regeneration. Examples of suchgrowth factors and their receptors is as follows: (G-CSF, FGFs, IGFs,KGF, NGF, VEGFs, PIGF, Angiopoietin, CTGF, PDGFs, HGF, EGF, HDGF,TGF-beta, Activins and Inhibins, Follistatin, BMPs, SCF/c-Kit, LIF,WNTs, SDFs, OncostatinM, Interleukins, Chemokines and many others);MMPs, TIMPs extracellular matrices (collagens, laminins, fibronectins,vitronectins, tenascins, intergrins, syndecans, decorin, fibromoludin,proteoglycans, sparc/osteonectin, mucin, netrin, glypican, cartilageassociated protein, matrilin, hyaluronan, fibulin, ADAMTS, biglycan,discoidin, desmosome components, ICAMs, cadherins, catenins and manyothers); cytokeratins.

There are groups of genes present only in UCMC. These genes are relatedto the following: Normal Physiological Processes (Insulin-like growthfactor 1 (somatomedin C); Insulin-like 4 (placenta); Relaxin 1;Plasminogen; Insulin-like growth factor 1 (somatomedin C); Insulin-like5; Insulin-like growth factor 1 (somatomedin C); Insulin-like growthfactor 2 (somatomedin A),Homeostasis (Radial spokehead-like 1;Hemochromatosis; Chemokine (C-C motif) ligand 5; Interleukin 31 receptorA; Chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1);Nuclear receptor subfamily 3, group C, member 2; Hemochromatosis;Chemokine (C-C motif) ligand 23; Chemokine (C-C motif) ligand 23;Ferritin mitochondrial; Peroxisome proliferative activated receptor,gamma, coactivator 1, alpha; Surfactant, pulmonary-associated protein D;Chemokine (C-C motif) ligand 11; Chemokine (C-C motif) ligand 3; Eglnine homolog 2 (C. elegans); Peroxisome proliferative activatedreceptor, gamma, coactivator 1, beta; Chemokine (C-C motif) ligand 1;Chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1);ATPase, Na+/K+ transporting, alpha 2 (+) polypeptide; Chemokine (Cmotif) ligand 2; Hemopexin; Ryanodine receptor 3), Morphogenesis(Spectrin, alpha, erythrocytic 1 (elliptocytosis 2); Homeo box D3; Eyesabsent homolog 1 (Drosophila); Ras homolog gene family, member J;Leukocyte specific transcript 1; Ectodysplasin A2 receptor; Glypican 3;Paired box gene 7; Corin, serine protease; Dishevelled, dsh homolog 1(Drosophila); Ras homolog gene family, member J; T-box 3 (ulnar mammarysyndrome); Chondroitin beta 1,4 N-acetylgalactosaminyltransferase;Chondroitin beta 1,4 N-acetylgalactosaminyltransferase; SRY (sexdetermining region Y)-box 10; Myosin, heavy polypeptide 9, non-muscle;Luteinizing hormone/choriogonadotropin receptor; radical fringe homolog(Drosophila); Secreted frizzled-related protein 5; Wingless-type MMTVintegration site family, member 11; Eyes absent homolog 2 (Drosophila);Muscleblind-like (Drosophila); T-box 5; Mab-21-like 1 (C. elegans);Growth arrest-specific 2; Sex comb on midleg homolog 1 (Drosophila);T-box 6; Filamin-binding LIM protein-1; Melanoma cell adhesion molecule;Twist homolog 1 (acrocephalosyndactyly 3; Saethre-Chotzen syndrome)(Drosophila); Homeo box A11; Keratocan; Fibroblast growth factor 1(acidic); Carboxypeptidase M; CDC42 effector protein (Rho GTPasebinding) 4; LIM homeobox transcription factor 1, beta; Engrailed homolog1; Carboxypeptidase M; Fibroblast growth factor 8 (androgen-induced);Fibroblast growth factor 18; Leukocyte specific transcript 1; Endothelin3; Paired-like homeodomain transcription factor 1), EmbryonicDevelopment (Pregnancy specific beta-1 -glycoprotein 3; ELAV (embryoniclethal, abnormal vision, Drosophila)-like 4 (Hu antigen D); Gprotein-coupled receptor 10; Ectodysplasin A2 receptor; ATP-bindingcassette, sub-family B (MDR/TAP), member 4; Pregnancy specific beta-1-glycoprotein 11; Nasal embryonic LHRH factor; Relaxin 1; Notch homolog4 (Drosophila); Pregnancy specific beta-1-glycoprotein 6; pih-2P; Homosapiens pregnancy-induced hypertension syndrome-related protein (PIH2);Oviductal glycoprotein 1, 120 kDa (mucin 9, oviductin);Progestagen-associated endometrial protein; Myosin, light polypeptide 4,alkali; atrial, embryonic; Prolactin; Notch homolog 4 (Drosophila);Pre-B-cell leukemia transcription factor 1; radical fringe homolog(Drosophila); Corticotropin releasing hormone; Nuclear receptorsubfamily 3, group C, member 2; Neuregulin 2; Muscleblind-like(Drosophila); Myosin, light polypeptide 4, alkali; atrial, embryonic;Homo sapiens cDNA FLJ27401 fis, clone WMCO3071; Extraembryonic,spermatogenesis, homeobox 1-like; Insulin-like 4 (placenta); Humanprocessed pseudo-pregnancy-specific glycoprotein (PSG12) gene, exon B2Ccontaining 3′ untranslated regions of 2 alternative splice sites C1 andC2; Fms-related tyrosine kinase 1 (vascular endothelial growthfactor/vascular permeability factor receptor); Pre-B-cell leukemiatranscription factor 1; Pregnancy specific beta-1-glycoprotein 3;carcinoembryonic antigen-related cell adhesion molecule 1 (biliaryglycoprotein); Steroid sulfatase (microsomal), arylsulfatase C, isozymeS; Homeo box B6; Protein O-fucosyltransferase 1; LIM homeoboxtranscription factor 1, beta; Carcinoembryonic antigen-related celladhesion molecule 1 (biliary glycoprotein); Follicle stimulatinghormone, beta polypeptide; Angiotensinogen (serine (or cysteine)proteinase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin),member 8); Carcinoembryonic antigen-related cell adhesion molecule 6(non-specific cross reacting antigen); Protein kinase C, alpha bindingprotein; Collectin sub-family member 10 (C-type lectin); Laminin, alpha1), the Extracellular Space (Carboxylesterase 1 (monocyte/macrophageserine esterase 1); Fibroblast growth factor 5; Progastricsin(pepsinogen C); Sperm associated antigen 11; Proprotein convertasesubtilisin/kexin type 2; Hyaluronan binding protein 2; Sema domain,immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin)3F; Interleukin 2; Chymotrypsin-like; Norrie disease (pseudoglioma);mucin 5, subtypes A and C, tracheobronchial/gastric; Carboxypeptidase B2(plasma, carboxypeptidase U); radical fringe homolog (Drosophila);Pregnancy specific beta-1-glycoprotein 11; Meprin A, alpha (PABA peptidehydrolase); Tachykinin, precursor 1 (substance K, substance P,neurokinin 1, neurokinin 2, neuromedin L, neurokinin alpha, neuropeptideK, neuropeptide gamma); Fibroblast growth factor 8 (androgen-induced);Fibroblast growth factor 13; Hemopexin; Breast cancer 2, early onset;Fibroblast growth factor 14; Retinoschisis (X-linked, juvenile) 1;Chitinase 3-like 1 (cartilage glycoprotein-39); Dystonin; Secretoglobin,family 1D, member 2; Noggin; WAP four-disulfide core domain 2; CD5antigen-like (scavenger receptor cysteine rich family); Scrapieresponsive protein 1; Gremlin 1 homolog, cysteine knot superfamily(Xenopus laevis); Interleukin 16 (lymphocyte chemoattractant factor);Chemokine (C-C motif) ligand 26; Nucleobindin 1; Fibroblast growthfactor 18; Insulin-like growth factor binding protein 1; Surfactant,pulmonary-associated protein A1; Delta-like 1 homolog (Drosophila);Cocaine- and amphetamine-regulated transcript; Meprin A, beta;Interleukin 17F; Complement factor H; Cysteine-rich secretory protein 2;Dystonin; WAP four-disulfide core domain 1; Prolactin; Surfactant,pulmonary-associated protein B; Fibroblast growth factor 5; Dickkopfhomolog 2 (Xenopus laevis); Sperm associated antigen 11; Chemokine (C-Cmotif) ligand 11; Meprin A, alpha (PABA peptide hydrolase); Chitinase3-like 2; C-fos induced growth factor (vascular endothelial growthfactor D); Chemokine (C-C motif) ligand 4; Poliovirus receptor;Hyaluronoglucosaminidase 1; Oviductal glycoprotein 1, 120 kDa (mucin 9,oviductin); Chemokine (C-X-C motif) ligand 9; Secreted frizzled-relatedprotein 5; Amelogenin (amelogenesis imperfecta 1, X-linked); Relaxin 1;Sparc/osteonectin, cwcv and kazal-like domains proteoglycan (testican);Chemokine (C-C motif) ligand 26; Fibroblast growth factor 1 (acidic);Angiopoietin-like 2; Fms-related tyrosine kinase 1 (vascular endothelialgrowth factor/vascular permeability factor receptor); Dystonin;Insulin-like 4 (placenta); Transcobalamin II; macrocytic anemia;Chemokine (C-C motif) ligand 1; Insulin-like growth factor bindingprotein, acid labile subunit; Complement factor H; Pregnancy specificbeta-1-glycoprotein 6; Silver homolog (mouse); Proteoglycan 4;Fibroblast growth factor 16; Cytokine-like protein C17; Granulysin;Angiopoietin 2; Chromogranin B (secretogranin 1); Sema domain,immunoglobulin domain (Ig), and GPI membrane anchor, (semaphorin) 7A;Pleiotrophin (heparin binding growth factor 8, neurite growth-promotingfactor 1); Chloride channel, calcium activated, family member 3;Secretoglobin, family 1 D, member 1; Fibulin 1; Phospholipase A2receptor 1, 180 kDa), and the Extracellular Matrix (ADAMTS-like 1;Periostin, osteoblast specific factor; Glypican 5; Leucine rich repeatneuronal 3; Transglutaminase 2 (C polypeptide,protein-glutamine-gamma-glutamyltransferase); A disintegrin-like andmetalloprotease (reprolysin type) with thrombospondin type 1 motif, 2;Microfibrillar-associated protein 4; Glypican 3; Collagen, type V, alpha3; Tissue inhibitor of metalloproteinase 2; Keratocan; Cartilageoligomeric matrix protein; Lumican; Hyaluronan and proteoglycan linkprotein 3; Statherin; A disintegrin-like and metalloprotease (reprolysintype) with thrombospondin type 1 motif, 3; Spondin 1, extracellularmatrix protein; Chitinase 3-like 1 (cartilage glycoprotein-39);Collagen, type IV, alpha 3 (Goodpasture antigen); Wingless-type MMTVintegration site family, member 7B; Collagen, type VI, alpha 2;Lipocalin 7; Hyaluronan and proteoglycan link protein 4; Adisintegrin-like and metalloprotease (reprolysin type) withthrombospondin type 1 motif, 5 (aggrecanase-2); Fibronectin 1; Matrilin1, cartilage matrix protein; Hypothetical protein FLJ13710; Chondroitinbeta 1,4 N-acetylgalactosaminyltransferase; Matrix metalloproteinase 16(membrane-inserted); Von Willebrand factor; Collagen, type VI, alpha 2;Transmembrane protease, serine 6; Matrix metalloproteinase 23B; Matrixmetalloproteinase 14 (membrane-inserted); Leucine rich repeat neuronal3; SPARC-like 1 (mast9, hevin); Sparc/osteonectin, cwcv and kazal-likedomains proteoglycan (testican) 3; Dermatopontin; collagen, type XIV,alpha 1 (undulin); Amelogenin, Y-linked; Nidogen (enactin); ADAMTS-like2; Hyaluronan and proteoglycan link protein 2; Collagen, type XV, alpha1; Glypican 6; Matrix metalloproteinase 12 (macrophage elastase);Amelogenin (amelogenesis imperfecta 1, X-linked); A disintegrin-like andmetalloprotease (reprolysin type) with thrombospondin type 1 motif, 15;Transmembrane protease, serine 6; A disintegrin-like and metalloprotease(reprolysin type) with thrombospondin type 1 motif, 16;Sparc/osteonectin, cwcv and kazal-like domains proteoglycan (testican);A disintegrin-like and metalloprotease (reprolysin type) withthrombospondin type 1 motif, 20; Collagen, type XI, alpha 1; Hyaluronanand proteoglycan link protein 1; Chondroitin beta 1,4N-acetylgalactosaminyltransferase; Asporin (LRR class 1); Collagen, typeIII, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal dominant);Secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, earlyT-lymphocyte activation 1); Matrix Gla protein; Fibulin 5; collagen,type XIV, alpha 1 (undulin); Tissue inhibitor of metalloproteinase 3(Sorsby fundus dystrophy, pseudoinflammatory); Collagen, type XXV, alpha1; Cartilage oligomeric matrix protein; Collagen, type VI, alpha 1;Chondroadherin; Collagen, type XV, alpha 1; A disintegrin-like andmetalloprotease (reprolysin type) with thrombospondin type 1 motif, 16;Collagen, type IV, alpha 4; Dentin matrix acidic phosphoprotein;Collagen, type IV, alpha 1; Thrombospondin repeat containing 1; Matrixmetalloproteinase 16 (membrane-inserted); Collagen, type I, alpha 2;Fibulin 1; Tectorin beta; Glycosylphosphatidylinositol specificphospholipase D1; Upregulated in colorectal cancer gene 1).Cytoskeleton: (Filamin B, beta (actin binding protein 278); Centrin,EF-hand protein, 1; FERM domain containing 3; Bridging integrator 3;Parvin, gamma; Rho guanine nucleotide exchange factor (GEF) 11; Tyrosinekinase 2; Kelch-like 4 (Drosophila); Spectrin, beta, erythrocytic(includes spherocytosis, clinical type I); Arg/Abl-interacting proteinArgBP2; Advillin; Spectrin repeat containing, nuclear envelope 1;Catenin (cadherin-associated protein), delta 1; Erythrocyte membraneprotein band 4.1 like 5; Catenin (cadherin-associated protein), alpha 2;Chemokine (C-C motif) ligand 3; Sarcoglycan, gamma (35kDadystrophin-associated glycoprotein); Nebulin; Thymosin, beta, identifiedin neuroblastoma cells; 3-phosphoinositide dependent protein kinase-1;Wiskott-Aldrich syndrome protein interacting protein; Dystonin;Huntingtin interacting protein 1; KIAA0316 gene product; Tropomodulin 4(muscle); Deleted in liver cancer 1; Villin-like; Syntrophin, beta 1(dystrophin-associated protein A1, 59 kDa, basic component 1); Proteinkinase, cGMP-dependent, type I; Homo sapiens similar to keratin 8;cytokeratin 8; keratin, type II cytoskeletal 8 (LOC345751), mRNA;Adducin 1 (alpha); Protein kinase C and casein kinase substrate inneurons 3; Dystonin; Kell blood group; Filamin A interacting protein 1;Growth arrest-specific 2; Chromosome 1 open reading frame 1;Stathmin-like 2; Spectrin, alpha, erythrocytic 1 (elliptocytosis 2);FKSG44 gene; Kinesin family member 1C; Tensin; Kaptin (actin bindingprotein); Neurofibromin 2 (bilateral acoustic neuroma); Pleckstrinhomology, Sec7 and coiled-coil domains 2 (cytohesin-2); Actin-relatedprotein T1; Wiskott-Aldrich syndrome-like; Kelch-like 4 (Drosophila);Fascin homolog 1, actin-bundling protein (Strongylocentrotuspurpuratus); Amphiphysin (Stiff-Man syndrome with breast cancer 128 kDaautoantigen); Polycystic kidney disease 2-like 1; Ankyrin 2, neuronal;CDC42 binding protein kinase alpha (DMPK-like); Hypothetical proteinFLJ36144; Arg/Abl-interacting protein ArgBP2; Formin-like 3; Catenin(cadherin-associated protein), beta 1, 88 kDa; Profilin 2; Synaptopodin2-like; Syntrophin, gamma 2; Phospholipase D2; Engulfment and cellmotility 2 (ced-12 homolog, C. elegans); Neurofilament, lightpolypeptide 68 kDa; Dystonin; Actin-like 7B; Kinesin family member 1C;PDZ and LIM domain 3; Adducin 2 (beta); obscurin, cytoskeletalcalmodulin and titin-interacting RhoGEF; Tubulin, beta polypeptideparalog; Filamin A interacting protein 1; Talin 1; Homo sapiens similarto [Segment 1 of 2] Piccolo protein (Aczonin) (LOC375597); CDC42effector protein (Rho GTPase binding) 4; Syndecan 1; Filamin A, alpha(actin binding protein 280); Profilin 2; Tensin like C1 domaincontaining phosphatase; Hypothetical protein MGC33407; Rho family GTPase1; Flavoprotein oxidoreductase MICAL2; Ca2+-dependent secretionactivator; Rabphilin 3A-like (without C2 domains); Myosin XVA; Proteinkinase, cGMP-dependent, type I; Myosin regulatory light chaininteracting protein; Kinesin family member 13B; Muscle RAS oncogenehomolog; Spectrin, beta, non-erythrocytic 1; TAO kinase 2; Filamin B,beta (actin binding protein 278); Neurofibromin 2 (bilateral acousticneuroma); Catenin (cadherin-associated protein), alpha 3; obscurin,cytoskeletal calmodulin and titin-interacting RhoGEF; Coronin, actinbinding protein, 1A; Erythrocyte membrane protein band 4.1-like 1;Spectrin, beta, non-erythrocytic 4; Thymosin, beta 4, Y-linked; Tektin 2(testicular); Ras homolog gene family, member J; Serine/threonine kinasewith Dbl- and pleckstrin homology domains; Dystrobrevin, beta; Actin,gamma 2, smooth muscle, enteric; Tara-like protein; Caspase 8,apoptosis-related cysteine protease; Kelch repeat and BTB (POZ) domaincontaining 10; Mucin 1, transmembrane; Microtubule-associated proteintau; Tensin; Ras homolog gene family, member F (in filopodia); Adducin 1(alpha); Actinin, alpha 4; Erythrocyte membrane protein band 4.1(elliptocytosis 1, RH-linked); Bicaudal D homolog 2 (Drosophila);Ankyrin 3, node of Ranvier (ankyrin G); Myosin VIIA (Usher syndrome 1B(autosomal recessive, severe)); Catenin (cadherin-associated protein),alpha 2; Homo sapiens similar to keratin 8, type II cytoskeletal-human(LOC285233); Fascin homolog 3, actin-bundling protein, testicular; Rashomolog gene family, member J; Beaded filament structural protein 2,phakinin; Desmin; Myosin X; Signal-induced proliferation-associated gene1; Scinderin; Coactosin-like 1 (Dictyostelium); Engulfment and cellmotility 2 (ced-12 homolog, C. elegans); Tubulin, beta 4; Ca²⁺-dependentsecretion activator; FERM domain containing 4A; Actin, alpha 1, skeletalmuscle; Talin 1; Caldesmon 1; Filamin-binding LIM protein-1;Microtubule-associated protein tau; Syntrophin, alpha 1(dystrophin-associated protein A1, 59 kDa, acidic component); Adducin 2(beta); Filamin A interacting protein 1; PDZ and LIM domain 3;Erythrocyte membrane protein band 4.1 like 4B; FYN binding protein(FYB-120/130); Bridging integrator 3). Extracellular: (Adisintegrin-like and metalloprotease (reprolysin type) withthrombospondin type 1 motif, 20; SPARC-like 1 (mast9, hevin); Serine (orcysteine) proteinase inhibitor, clade G (C1 inhibitor), member 1,(angioedema, hereditary); Urocortin; Chymotrypsin-like; Platelet-derivedgrowth factor beta polypeptide (simian sarcoma viral (v-sis) oncogenehomolog); BMP-binding endothelial regulator precursor protein;Complement factor H; Chorionic somatomammotropin hormone-like 1;Chemokine (C-C motif) ligand 18 (pulmonary and activation-regulated);Fibronectin 1; Pregnancy specific beta-1-glycoprotein 3; Adisintegrin-like and metalloprotease (reprolysin type) withthrombospondin type 1 motif, 3; CocoaCrisp; Insulin-like 4 (placenta);Wingless-type MMTV integration site family, member 11; Cartilageoligomeric matrix protein; Transmembrane protease, serine 6; C-fosinduced growth factor (vascular endothelial growth factor D); Familywith sequence similarity 12, member B (epididymal); Protein phosphatase1, regulatory subunit 9B, spinophilin; Transcobalamin II; macrocyticanemia; Coagulation factor V (proaccelerin, labile factor);Phospholipase A2, group IID; Tumor necrosis factor, alpha-inducedprotein 6; Collagen, type XV, alpha 1; Hyaluronan and proteoglycan linkprotein 3; collagen, type XIV, alpha 1 (undulin) ; Interleukin 19;Protease inhibitor 15; Cholinergic receptor, nicotinic, beta polypeptide1 (muscle); Lysyl oxidase-like 3; Insulin-like growth factor bindingprotein 5; Growth hormone 1; Casein beta; NEL-like 2 (chicken); I factor(complement); Chemokine (C-C motif) ligand 23; Interferon, alpha 2;Matrix metalloproteinase 16 (membrane-inserted); Matrixmetalloproteinase 12 (macrophage elastase); Glypican 5; Pregnancyspecific beta-1-glycoprotein 3; Fibroblast growth factor 6; Gremlin 1homolog, cysteine knot superfamily (Xenopus laevis); Protein S (alpha);Chondroitin beta 1,4 N-acetylgalactosam inyltransferase;Glycosylphosphatidylinositol specific phospholipase D1; Fibroblastgrowth factor 1 (acidic); Spondin 1, extracellular matrix protein; Bonemorphogenetic protein 1; Surfactant, pulmonary-associated protein B;Dentin matrix acidic phosphoprotein; Lipoprotein, Lp(a); Mucin 1,transmembrane; Mannan-binding lectin serine protease 1 (C4/C2 activatingcomponent of Ra-reactive factor); Meprin A, beta; Secretoglobin, family1D, member 1; Asporin (LRR class 1); Chemokine (C-C motif) ligand 25;Cytokine-like protein C17; Insulin-like 5; Meprin A, alpha (PABA peptidehydrolase); Scrapie responsive protein 1; Fibroblast growth factor 18;Chemokine (C-X-C motif) ligand 9; Inhibin, beta B (activin AB betapolypeptide); Fibroblast growth factor 8 (androgen-induced); Granulysin;Cocaine- and amphetamine-regulated transcript; Collagen, type I, alpha2; Chemokine (C-C motif) ligand 17; Chemokine (C-C motif) ligand 23;Sparc/osteonectin, cwcv and kazal-like domains proteoglycan (testican)3; Gamma-aminobutyric acid (GABA) A receptor, beta 3; Defensin, alpha 4,corticostatin; Leucine rich repeat neuronal 3; Glypican 6;Mitogen-activated protein kinase kinase 2; Coagulation factor XI (plasmathromboplastin antecedent); Chemokine (C-C motif) ligand 5; Dystonin;Frizzled-related protein; Coagulation factor XIII, A1 polypeptide;Insulin-like growth factor 1 (somatomedin C); Hypothetical proteinMGC45438; Sperm associated antigen 11; Insulin-like growth factor 1(somatomedin C); Periostin, osteoblast specific factor;Alpha-2-macroglobulin; Gamma-aminobutyric acid (GABA) A receptor, alpha5; Serine (or cysteine) proteinase inhibitor, clade A (alpha-1antiproteinase, antitrypsin), member 3; Silver homolog (mouse);Frizzled-related protein; Chondroadherin; Chondroitin beta1,4N-acetylgalactosam inyltransferase; 5-hydroxytryptamine (serotonin)receptor 3, family member C; Collagen, type VI, alpha 2; Toll-likereceptor 9; Amelogenin, Y-linked; Vascular endothelial growth factor B;Radial spokehead-like 1; Fms-related tyrosine kinase 1 (vascularendothelial growth factor/vascular permeability factor receptor);Protease inhibitor 16; Interleukin 2; Clusterin (complement lysisinhibitor, SP-40,40, sulfated glycoprotein 2, testosterone-repressedprostate message 2, apolipoprotein J); Follicle stimulating hormone,beta polypeptide; A disintegrin-like and metalloprotease (reprolysintype) with thrombospondin type 1 motif, 16; Lysozyme (renalamyloidosis); radical fringe homolog (Drosophila); Insulin-like growthfactor binding protein 5; Taxilin; Apolipoprotein A-V; Platelet derivedgrowth factor C; Chemokine (C-C motif) ligand 3-like 1; Fibroblastgrowth factor 16; Collagen, type VI, alpha 2; Serine (or cysteine)proteinase inhibitor, clade C (antithrombin), member 1; Chemokine (C-Cmotif) ligand 11; Collagen, type IV, alpha 4; Bruton agammaglobulinemiatyrosine kinase; Insulin-like growth factor 2 (somatomedin A);Kazal-type serine protease inhibitor domain 1; Fibrinogen, A alphapolypeptide; Chemokine (C-C motif) ligand 1; Inhibin, beta E; Sexhormone-binding globulin; Collagen, type IV, alpha 1;Lecithin-cholesterol acyltransferase; Cysteine-rich secretory protein 2;Hyaluronan and proteoglycan link protein 1; Natriuretic peptideprecursor C; Ribonuclease, RNase A family, k6; Fibroblast growth factor14; ADAMTS-like 2; Collagen, type IV, alpha 3 (Goodpasture antigen);Angiopoietin 2; Apolipoprotein L, 3; Chemokine (C-X-C motif) ligand 12(stromal cell-derived factor 1); Hyaluronan binding protein 2;Coagulation factor VII (serum prothrombin conversion accelerator);collagen, type XIV, alpha 1 (undulin); Oviductal glycoprotein 1, 120 kDa(mucin 9, oviductin); Matrilin 1, cartilage matrix protein; mucin 5,subtypes A and C, tracheobronchial/gastric; Tumor necrosis factorreceptor superfamily, member 11b (osteoprotegerin); Transglutaminase 2(C polypeptide, protein-glutamine-gamma-glutamyltransferase); Keratocan;Collagen, type V, alpha 3; WAP four-disulfide core domain 2; Chemokine(C-X3-C motif) ligand 1; Serine (or cysteine) proteinase inhibitor,clade D (heparin cofactor), member 1; Secretory protein LOC348174;Coagulation factor X; Interleukin 16 (lymphocyte chemoattractantfactor); Pancreatic lipase-related protein 2; HtrA serine peptidase 3;Glycine receptor, alpha 3; CD5 antigen-like (scavenger receptor cysteinerich family); Hypothetical protein MGC39497; Coagulation factor VIII,procoagulant component (hemophilia A); Dermatopontin; Noggin; SecretedLY6/PLAUR domain containing 1; ADAMTS-like 1; Alpha-1-B glycoprotein;Chromosome 20 open reading frame 175; Wingless-type MMTV integrationsite family, member 8B; Fibulin 1; Fibulin 5; Cathepsin S; Nidogen(enactin); Chemokine (C-C motif) ligand 26; Endothelial cell-specificmolecule 1; Chitinase 3-like 1 (cartilage glycoprotein-39);Gamma-aminobutyric acid (GABA) A receptor, beta 1; Secretoglobin, family1D, member 2; Mannan-binding lectin serine protease 1 (C4/C2 activatingcomponent of Ra-reactive factor); ADAMTS-like 1; Sema domain,immunoglobulin domain (Ig), and GPI membrane anchor, (semaphorin) 7A; Adisintegrin-like and metalloprotease (reprolysin type) withthrombospondin type 1 motif, 15; Proprotein convertase subtilisin/kexintype 2; Insulin-like growth factor 1 (somatomedin C); Retinoschisis(X-linked, juvenile) 1; A disintegrin-like and metalloprotease(reprolysin type) with thrombospondin type 1 motif, 16; Chemokine (Cmotif) ligand 2; Fibroblast growth factor 5; Sperm associated antigen11; Microfibrillar-associated protein 4; Poliovirus receptor;Extracellular signal-regulated kinase 8; Transmembrane protease, serine6; Protein kinase C, alpha; Chitinase 3-like 2; Interleukin 9;Apolipoprotein L, 6; Surfactant, pulmonary-associated protein A1;Collagen, type VI, alpha 1; Apolipoprotein L, 6; Hypothetical proteinFLJ13710; Carboxypeptidase B2 (plasma, carboxypeptidase U) ;Bactericidal/permeability-increasing protein-like 2; Fibroblast growthfactor 5; Secreted phosphoprotein 1 (osteopontin, bone sialoprotein I,early T-lymphocyte activation 1); HtrA serine peptidase 3; Deleted inliver cancer 1; Endothelial cell-specific molecule 1; Von Willebrandfactor; A disintegrin-like and metalloprotease (reprolysin type) withthrombospondin type 1 motif, 5 (aggrecanase-2); Sema domain,immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin)3A; Chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1);Statherin; Extracellular signal-regulated kinase 8; Tissue inhibitor ofmetalloproteinase 3 (Sorsby fundus dystrophy, pseudoinflammatory);Platelet factor 4 (chemokine (C-X-C motif) ligand 4); Surfactant,pulmonary-associated protein D; Complement factor H; Delta-like 1homolog (Drosophila); WAP four-disulfide core domain 1; Insulin-likegrowth factor binding protein, acid labile subunit; Breast cancer 2,early onset; Pre-B lymphocyte gene 1; Corticotropin releasing hormone;Hypothetical protein DKFZp434B044; Prolactin-induced protein; RAS guanylreleasing protein 4; Progastricsin (pepsinogen C); Sema domain,immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin)3F; Upregulated in colorectal cancer gene 1; Proteoglycan 4; Cholinergicreceptor, nicotinic, delta polypeptide; Cartilage oligomeric matrixprotein; ABO blood group (transferase A, alpha1-3-N-acetylgalactosaminyltransferase; transferase B, alpha1-3-galactosyltransferase); Interleukin 12A (natural killer cellstimulatory factor 1, cytotoxic lymphocyte maturation factor 1, p35);Fibroblast growth factor 7 (keratinocyte growth factor); Kin of IRRElike 3 (Drosophila); Cholinergic receptor, nicotinic, alpha polypeptide2 (neuronal); Palate, lung and nasal epithelium carcinoma associated;Collagen, type XV, alpha 1; Pleiotrophin (heparin binding growth factor8, neurite growth-promoting factor 1); Angiopoietin-like 2; Norriedisease (pseudoglioma); Chemokine (C-C motif) ligand 3; Chitinase 3-like1 (cartilage glycoprotein-39); Inter-alpha (globulin) inhibitor H3;Amelogenin (amelogenesis imperfecta 1, X-linked); Epidermal growthfactor (beta-urogastrone); Fibroblast growth factor 13; Wingless-typeMMTV integration site family, member 7B; Cholinergic receptor,nicotinic, gamma polypeptide; Pregnancy specific beta-1-glycoprotein 6;Matrix metalloproteinase 14 (membrane-inserted); Chemokine (C-C motif)ligand 26; Interferon, alpha 6; Tachykinin, precursor 1 (substance K,substance P, neurokinin 1, neurokinin 2, neuromedin L, neurokinin alpha,neuropeptide K, neuropeptide gamma); Secreted frizzled-related protein5; Hyaluronan and proteoglycan link protein 4; Complement component 4B;Matrix metalloproteinase 16 (membrane-inserted); Fibroblast growthfactor 7 (keratinocyte growth factor); Apolipoprotein C-II; Chloridechannel, calcium activated, family member 3; Tetranectin (plasminogenbinding protein); Collagen, type III, alpha 1 (Ehlers-Danlos syndrometype IV, autosomal dominant); KIAA0556 protein; Chemokine (C-C motif)ligand 4; Hemopexin; Inter-alpha (globulin) inhibitor H1; Relaxin 1;Matrix Gla protein; A disintegrin-like and metalloprotease (reprolysintype) with thrombospondin type 1 motif, 2; Interferon (alpha, beta andomega) receptor 2; Acid phosphatase, prostate; Guanine nucleotidebinding protein (G protein), gamma 8; Matrix metalloproteinase 23B;Meprin A, alpha (PABA peptide hydrolase); Hyaluronoglucosaminidase 1;Angiotensinogen (serine (or cysteine) proteinase inhibitor, clade A(alpha-1 antiproteinase, antitrypsin), member 8); Cartilage intermediatelayer protein, nucleotide pyrophosphohydrolase; Purinergic receptor P2X,ligand-gated ion channel, 7; Glypican 3; Tectorin beta; Interferon,alpha 5; Lipocalin 7; Platelet factor 4 variant 1; Nucleobindin 1;Collagen, type XI, alpha 1; Gastric inhibitory polypeptide;Thrombospondin repeat containing 1; 5-hydroxytryptamine (serotonin)receptor 3 family member D; Collagen, type XXV, alpha 1; Growthdifferentiation factor 9; Hypothetical protein DKFZp434B044; Endothelin3; Chemokine (C motif) ligand 2; Prokineticin 2; Tumor necrosis factorreceptor superfamily, member 11b (osteoprotegerin); Tissue inhibitor ofmetalloproteinase 2; Dystonin; Chromogranin B (secretogranin 1);Hyaluronan and proteoglycan link protein 2; Leucine rich repeat neuronal3; Lumican; Matrilin 1, cartilage matrix protein; Phospholipase A2,group IIA (platelets, synovial fluid); Carboxylesterase 1(monocyte/macrophage serine esterase 1); Sparc/osteonectin, cwcv andkazal-like domains proteoglycan (testican); Dickkopf homolog 2 (Xenopuslaevis); Gamma-aminobutyric acid (GABA) A receptor, alpha 3; Pregnancyspecific beta-1 -glycoprotein 11; Insulin-like growth factor bindingprotein 1; Defensin, beta 106; Interleukin 17F; Ligand-gated ion channelsubunit ; Phospholipase A2 receptor 1, 180 kDa; I factor (complement);Dystonin; LAG1 longevity assurance homolog 1 (S. cerevisiae); Prolactin;Testis expressed sequence 264; Sema domain, immunoglobulin domain (Ig),short basic domain, secreted, (semaphorin) 3D; secreted frizzled-relatedprotein 2; secreted frizzled-related protein 4).

There are groups of genes present only in UCEC. These genes are relatedto the following: Homeostasis (Albumin; Calcium-sensing receptor;Aquaporin 9; Lactotransferrin. Morphogenesis: Homeo box HB9; EpithelialV-like antigen 1). Embryonic Development (Relaxin 2; Carcinoembryonicantigen-related cell adhesion molecule 8; Indoleamine-pyrrole 2,3dioxygenase; EPH receptor A3; Thyrotrophic embryonic factor; Pregnancyspecific beta-1-glycoprotein 1; Laminin, alpha 3), the ExtracellularSpace (Surfactant, pulmonary-associated protein A1; Pregnancy specificbeta-1 -glycoprotein 1; Lactotransferrin; TGF-alpha; Albumin; FGF-23;S100 calcium binding protein A9 (calgranulin B)), the ExtracellularMatrix (Laminin, beta 4; Laminin, alpha 3; Zona pellucida glycoprotein4. Structural Molecule Activity: Chromosome 21 open reading frame 29;Laminin, alpha 3; Microtubule-associated protein 2; Laminin, beta 4;Keratin 6B; Ladinin 1; Keratin 6A; Occludin; Loricrin; Erythrocytemembrane protein band 4.1 (elliptocytosis 1, RH-linked); Crystallin,beta A2; eye lens structural protein; Contactin associated protein-like4; Claudin 19; Hypothetical protein LOC144501; Keratin 6E; Keratin 6L;Lens intrinsic membrane protein 2, 19 kDa), the Cytoskeleton(Microtubule-associated protein 2; Erythrocyte membrane protein band 4.1like 5; Homo sapiens trichohyalin (THH); Keratin 6B; Keratin 6A;Epithelial V-like antigen 1; Hook homolog 1 (Drosophila); Loricrin;Erythrocyte membrane protein band 4.1 (elliptocytosis 1, RH-linked);Tropomodulin 1; MAP/microtubule affinity-regulating kinase 1; Keratin6E; Actin binding LIM protein family, member 2), Cell Adhesion Molecules(Cadherin 19, type 2; Myeloid/lymphoid or mixed-lineage leukemia;Chromosome 21 open reading frame 29; Kin of IRRE like 2; Laminin, alpha3; Sialoadhesin; CD84 antigen (leukocyte antigen); Lectin,galactoside-binding, soluble, 2 (galectin 2); Epithelial V-like antigen1; CD96 antigen; Tubulointerstitial nephritis antigen; Carcinoembryonicantigen-related cell adhesion molecule 8; IL-18; Immunoglobulinsuperfamily, member 1; Integrin, beta 8; Ornithine arbamoyltransferase;Integrin, beta 6; Contactin associated protein-like 4; Collagen, typeXVII, alpha 1; Cadherin-like 26; Mucin and cadherin-like), CellDifferentiation proteins (Protein tyrosine phosphatase, receptor-type, Zpolypeptide 1; Laminin, alpha 3; CD84 antigen (leukocyte antigen);EDRF2; Homo sapiens erythroid differentiation-related factor 2; Tumorprotein p73-like; NB4 apoptosis/differentiation related protein; Homosapiens PNAS-133; Similar to seven in absentia 2; Interleukin 24;Keratin 6B; Keratin 6A; Dehydrogenase/reductase (SDR family) member 9;Gap junction protein, beta 5 (connexin 31.1); Iroquois homeobox protein4; Ventral anterior homeobox 2; Chemokine (C-X-C motif) ligand 10; Tumornecrosis factor receptor superfamily, member 17; Calcium channel,voltage-dependent, beta 2 subunit; Parkinson disease (autosomalrecessive, juvenile) 2, parkin; Kallikrein 7 (chymotryptic, stratumcorneum); Glial cells missing homolog 2; AP-2 alpha; Protein tyrosinephosphatase, receptor-type, Z polypeptide 1; Troponin T1; Sciellin;Glucosaminyl (N-acetyl) transferase 2, I-branching enzyme; Collagen,type XVII, alpha 1; Suppressor of cytokine signaling 2; Distal-lesshomeo box 1; Zygote arrest 1; Interleukin 20; Growth differentiationfactor 3; FGF-23; Wingless-type MMTV integration site family, member 8A.Extracellular: Chromosome 21 open reading frame 29; Laminin, alpha 3;Laminin, beta 4; Interleukin 24; Pregnancy specific beta-1 -glycoprotein1; Chemokine (C-X-C motif) ligand 11; Surfactant, pulmonary-associatedprotein A1; Prepronociceptin; 5-hydroxytryptamine (serotonin) receptor3B; Carcinoembryonic antigen-related cell adhesion molecule 8; Chemokine(C-X-C motif) ligand 10; IL-18 (interferon-gamma-inducing factor);Lactotransferrin; Albumin; Fas ligand (TNF superfamily, member 6);Cholinergic receptor, nicotinic, beta polypeptide 4; Cathelicidinantimicrobial peptide; Airway trypsin-like protease;S100 calcium bindingprotein A9 (calgranulin B); TGF-alpha; Kallikrein 10; Serine proteaseinhibitor, Kunitz type 1; WNT1 inducible signaling pathway protein 3;Relaxin 2; Interferon, kappa; Defensin, beta 103A; IL-20; Zona pellucidaglycoprotein 4; Growth differentiation factor 3; FGF-23; Wingless-typeMMTV integration site family, member 8A; Complement factor H-related 5),Developmental proteins (EPH receptor A3; NIMA (never in mitosis genea)-related kinase 2; Zinc finger protein 282; TANK-binding kinase 1;MRE11 meiotic recombination 11 homolog A; E2F transcription factor 2;Protein tyrosine phosphatase, receptor-type, Z polypeptide 1; Homosapiens clone 161455 breast expressed mRNA from chromosome X; Laminin,alpha 3; v-myb myeloblastosis viral oncogene homolog (avian)-like 1;Regulator of G-protein signalling 11; Microtubule-associated protein 2;Transmembrane protein 16A; Adenomatosis polyposis coli 2; Homeo box HB9;Centromere protein F, 350/400 ka (mitosin); CD84 antigen (leukocyteantigen); EDRF2; Homo sapiens erythroid differentiation-related factor2; Tumor protein p73-like; NB4 apoptosis/differentiation relatedprotein; Homo sapiens PNAS-133; Forkhead box P2; Homo sapiensgastric-associated differentially-expressed protein YA61P (YA61);Tenascin N; Chromosome 6 open reading frame 49; Zinc finger protein 462;Zinc finger protein 71 (Cos26); SRY (sex determining region Y)-box 7;Triggering receptor expressed on myeloid cells-like 4; Interleukin 24;Pregnancy specific beta-1-glycoprotein 1; Chondroitin sulfateproteoglycan 5 (neuroglycan C); Keratin 6B; Keratin 6A;Dehydrogenase/reductase (SDR family) member 9; Epithelial V-like antigen1; Gap junction protein, beta 5 (connexin 31.1); G protein-coupledreceptor 51; Interferon regulatory factor 6; Neurotrophin 5(neurotrophin 4/5); CD96 antigen; Iroquois homeobox protein 4;Interleukin 1 receptor-like 1; G-2 and S-phase expressed 1; Nuclearreceptor subfamily 2, group E, member 3; Ventral anterior homeobox 2;Zinc finger protein 215; DNA segment on chromosome 4 (unique) 234expressed sequence; Carcinoembryonic antigen-related cell adhesionmolecule 8; Chemokine (C-X-C motif) ligand 10; IL-18;Indoleamine-pyrrole 2,3 dioxygenase; Albumin; Calcium-sensing receptor(hypocalciuric hypercalcemia 1, severe neonatal hyperparathyroidism);Fas ligand (TNF superfamily, member 6); TNFR superfamily, member 17;Calcium channel, voltage-dependent, beta 2 subunit; Parkinson disease(autosomal recessive, juvenile) 2, parkin; Kallikrein 7 (chymotryptic,stratum corneum); Glial cells missing homolog 2; TGF-alpha; Thyrotrophicembryonic factor; AP-2 alpha (activating enhancer binding protein 2alpha); Kallikrein 10; Regulator of G-protein signalling 7; Proteintyrosine phosphatase, receptor-type, Z polypeptide 1; Serine proteaseinhibitor, Kunitz type 1; WNT1 inducible signaling pathway protein 3;Zic family member 3 heterotaxy 1 (odd-paired homolog, Drosophila); TTKprotein kinase; Troponin T1, skeletal, slow; Sciellin; TGFB-inducedfactor 2-like, X-linked; Kallikrein 8 (neuropsin/ovasin); Glucosaminyl(N-acetyl) transferase 2, I-branching enzyme; Ankyrin repeat domain 30A;Relaxin 2; Collagen, type XVII, alpha 1; Gene differentially expressedin prostate; Phosphatase and actin regulator 3; Suppressor of cytokinesignaling 2; Nuclear receptor subfamily 4, group A, member 3;Angiotensin I converting enzyme (peptidyl-dipeptidase A) 1; Hypotheticalprotein MGC17986; Distal-less homeo box 1; LAG1 longevity assurancehomolog 3 (S. cerevisiae); Zygote arrest 1; Interferon, kappa; IL-20;ICEBERG caspase-1 inhibitor; Growth differentiation factor 3; FGF-23;Testis expressed sequence 15; Wingless-type MMTV integration sitefamily, member 8A; SRY (sex determining region Y)-box 7; Carnitinedeficiency-associated, expressed in ventricle 1; Prokineticin 1; CAMPresponsive element binding protein 3-like 3; Caspase recruitment domainfamily, member 15; FLJ23311 protein).

Example 6 Direct Differentiation of Umbilical Cord Epithelial Stem Cells(UCEC) into Skin Epidermal Keratinocytes

For differentiation into skin epidermal keratinocytes, umbilical cordepithelial stem cells, UCEC cells, were cultured according to a standardprotocol for the cultivation of keratinocytes. Cell isolation techniqueswere as described above. UCEC were then cultured in serum-freekeratinocyte growth media, KGM, KGM-2 (Cambrex), EpiLife (CascadeBiologics) or in Green's medium in the presence of irradiated orMytomycin-C treated 3T3 mouse embryonic feeder layer at 37° C., 5% CO₂).UCEC cell morphology thus differentiated resembled human epidermalkeratinocytes. Epithelial cells have similar morphology under lightmicroscope and can be easily turned into fibroblasts using conventionaland commercially available media (cf., FIG. 2).

Immunofluorescent analysis shows that the cultivated UCEC also expressepidermal keratinocyte molecular markers such as keratins, desmosome,hemidesmosome and basement membrane components (see also FIG. 10 thatshows that UCEC are qualified to be epithelial cells in general byexpressing a variety of these epithelial cell markers). Accordingly,these results show that umbilical cord epithelial progenitor/stem cellsof the present invention can be differentiated into skin cells such asepidermal keratinocytes which can be used for wound healing and havegreat potential for the development of cultured skin equivalents.

Example 7 Expansion of Umbilical Cord Epithelial and Mesenchymal StemCells Using Repetitive Tissue Explants of Umbilical Cord Lining MembraneTissues

Umbilical cord epithelial and mesenchymal stem cells of the inventionwere expanded using repetitive explants of umbilical cord amnioticmembrane tissue as follows. Briefly, at day 1 of process, tissueexplants were plated onto tissue culture dishes in growth media(DMEM/10% FCS, EpiLife, KGM, KGM-2 or M171) at 37° C., 5% CO₂; media waschanged every 2 or 3 days. Cell outgrowths started and continuedmigrating from the explants for 7 days. After that, tissue explants weretransferred to other dishes to allow further cell outgrowth. Thisprocess was continued until the explants had diminished in size,preventing further explantation. In this connection it is noted that theexplants progressively shrink in size until they are too small forfurther tissue explant since during the process of cells outgrowing andmigrating from tissue explants, the cells produce proteases to digestand break down tissue. FIG. 16 schematically illustrates the rapid androbust expansion process of umbilical cord epithelial and mesenchymalstem cells achieved using this protocol. Thus, this study demonstratesthe high yield of UCMC and UMEC cells can be obtained from this source,further reflecting the high viability and pro-growth characteristics oftthese cells in comparison with other sources of cells as bone-marrow oradipose-derived stem cells. In addition, being a solid tissue, thesuccessful repetitive explant technique used herein demonstrates thatthe cells of the invention can be uniformly extracted from the entiretissue instead of only certain portions. This allows the maximum numberof cells that can be derived at a low passage instead of passing thecells through many generations causing deterioration of cells.

Example 8 Direct Differentiation of Umbilical Cord Mesenchymal Cells(UCMC) into Skin Dermal Fibroblasts

For differentiation into skin dermal fibroblasts, umbilical cordmesenchymal stem cells, UCMC cells were cultured according to a standardprotocol for the cultivation of fibroblasts. Cell isolation techniqueswere as described above in Example 6. UCMC were then cultured in DMEM orcommercially available fibroblast growth media (FGM). UCMC cellmorphology thus differentiated resembled human dermal fibroblasts.Mesenchymal cells have similar morphology under light microscope and canbe easily turned into fibroblasts using conventional and commerciallyavailable media (cf., FIG. 3).

Example 9 Direct Differentiation of Epithelial Stem/Progenitor Cellsinto Skin Epidermal Keratinocytes

In an approach similar to Example 6, epithelial stem/progenitor cells ofthe amniotic membrane of the umbilical cord (UCEC) were isolated asdescribed in Example 2. For differentiation of UCEC into epidermalkeratinocytes, the cells were cultured in keratinocyte media (EpiLife orKGM) until 100% (cultivation after 5 days shown in FIG. 17-A) confluentbefore changing the media to DMEM/10% FCS for 3 days to form epidermalcell sheets. As shown in FIG. 17-A (in which photographs of twoexperiments termed “UCEC-10” and UCEC-17 are depicted), aftercultivation in DMEM/10% FCS, UCEC, had differentiated into epidermalkeratinocytes that formed cell sheets (photograph of FIG. 17-A takenafter 10 days). These results thus provide further evidence for thepluripotency of the cells of the present invention.

Example 10 Direct Differentiation of Mesenchymal Stem/Progenitor Cellsinto Osteoblasts

Mesenchymal stem/progenitor cells of the amniotic membrane of theumbilical cord (UCMC) were isolated as described in Example 2. Fordifferentiation of UCMC into osteoblasts, cells were cultured inDMEM/10% FCS until 100% confluent, and then in starvation medium ofserum-free DMEM for another 48 hours. UCMC were subjected to osteogenicinduction media for 4 weeks before subjecting the cells to von Kossastaining (bone cell staining). The osteogenic induction medium containedDMEM/10% FCS; 1% antibiotic (streptomycin and penicillin)/antimycotic(fungizone); 0.01 μM 1,25-dihydroxyvitamin D3, 50 μMascorbate-2-phosphate, 10 mM β-glycerophosphate, 1% antibiotic(streptomycin and penicillin)/antimycotic (fungizone).

As shown in FIG. 17B, von Kossa staining of UCMC cells that werecultivated in the osteogenic induction medium indicated bone noduleformation in the UCMC and thus differentiation of the UCMC intoosteoblasts whereas no such differentiation was indicated in untreatedUCMC which were cultured in DMEM/10% FCS without induction underotherwise same conditions as negative control. As a further negativecontrol, dermal fibroblasts from an 8 months old donor and keloidfibroblasts from an 20 year old donor were cultivated under the sameconditions as the induced or un-induced UCMC. Both cell types did notyield a positive result using von Kossa staining, which is a furtherevidence for the pluripotency of UCMC of the present invention and thusto differentiate, for example, also into osteoblasts.

Example 11 Direct Differentiation of Mesenchymal Stem/Progenitor Cellsinto Adipocytes

Mesenchymal stem/progenitor cells from the amniotic membrane of theumbilical cord (UCMC) were isolated as described in Example 2. Fordifferentiation of UCMC into adipocytes, cells were cultured in DMEM/10%FCS until 100% confluent, and then in starvation medium of serum-freeDMEM for another 48 hours. UCMC were subjected to adipogenic inductionmedia for 4 weeks before subjecting the cells to Oil-Red-O staining. Theadipogenic induction medium contained DMEM/10%FCS; 1% antibiotic(streptomycin and penicillin)/antimycotic (fungizone)); 0.5 mMisobutyl-methylxanthine (IBMX), 1 μM dexamethasone, 10 μM insulin, and200 μM indomethacin.

Oil-Red-O staining of UCMC cells that were cultivated in the adipogenicinduction medium indicated fat accumulation in the UCMC and thusdifferentiation of the UCMC into adipocytes whereas no suchdifferentiation was indicated in untreated UCMC which were cultured inDMEM/10% FCS without induction under otherwise same conditions asnegative control. As a further negative control, dermal fibroblasts froman 8 month old donor and keloid fibroblasts from a 20 year old donorwere cultivated under the same conditions as the induced or un-inducedUCMC. Both cell types did not yield a positive result in the stainingwith Oil-Red-O, which is a further evidence for the pluripotency of UCMCof the present invention and to differentiate, for example, also intoadipocytes.

What is claimed is:
 1. A method for isolating stem/progenitor cells fromthe amniotic membrane of an umbilical cord, the method comprising: (a)separating the amniotic membrane from the other components of theumbilical cord in vitro to obtain an amniotic membrane tissue; (b)culturing the amniotic membrane tissue obtained in step (a) underconditions allowing cell proliferation; and (c) isolating thestem/progenitor cells, wherein the stem/progenitor cells are epithelialor mensenchymal stem/progenitor cells expressing the following genes:POU5fl, Bmi-1, leukemia inhibitory factor (LIF), and secreting Activin Aand follistatin.
 2. The method of claim 1, further comprising: (a″)separating the cells of the amniotic membrane tissue before cultivationby a method selected from the group consisting of enzymatic separationand direct tissue explant.
 3. The method of claim 1, further comprising:(d) culturing the stem/progenitor cells under conditions allowing thecells to undergo clonal expansion.
 4. The method of claim 3, furthercomprising: (e) culturing the stem/progenitor cells under conditionsallowing the differentiation of said cells into epithelial cells ormesenchymal cells; and (f) isolating the differentiated cells.
 5. Themethod of claim 4, wherein the epithelial cells are skin epidermalkeratinocytes.
 6. The method of claim 4, wherein the mesenchymal cellsare selected from the group consisting of skin fibroblasts, osteoblastsand adipocytes.
 7. The method of claim 1, further comprising (g)preserving the isolated stem/progenitor cells for further use.
 8. Themethod of claim 7, wherein preserving is carried out by usingcryo-preservation.
 9. A method of differentiating a stem/progenitor cellisolated by the method as defined in claim 1, comprising culturing thestem/progenitor cells under conditions allowing the cells to undergoclonal expansion.
 10. The method of claim 9, further comprisingculturing the stem/progenitor cells under conditions allowing thedifferentiation of said cells into epithelial cells and/or mesenchymalcells.
 11. The method of claim 10, wherein the epithelial cells are skinepidermal keratinocytes.
 12. The method of claim 10, wherein themesenchymal cells are selected from the group consisting of skinfibroblasts, osteoblasts, and adipocytes.